Methods of treating FGF21-associated disorders

ABSTRACT

The present invention relates to monoclonal antibodies and antigen-binding fragments thereof that bind to human β-klotho, and pharmaceutical compositions and methods of treatment comprising the same.

This application claims the benefit of U.S. Provisional Application No.62/200,445 filed on Aug. 3, 2015, which is hereby incorporated byreference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 28, 2016, isnamed PAT056954_SL.txt and is 124,487 bytes in size.

FIELD

The present invention relates to fibroblast growth factor 21 (FGF21)mimetic antibodies. Also disclosed are methods for treatingFGF21-associated disorders, such as obesity, type 1 and type 2 diabetesmellitus, pancreatitis, dyslipidemia, nonalcoholic steatohepatitis(NASH), insulin resistance, hyperinsulinemia, glucose intolerance,hyperglycemia, metabolic syndrome, and other metabolic disorders, and inreducing the mortality and morbidity of critically ill patients.

BACKGROUND

The fibroblast growth factor (FGF) family is characterized by 22genetically distinct, homologous ligands, which are grouped into sevensubfamilies. According to the published literature, the FGF family nowconsists of at least twenty-three members, FGF-1 to FGF-23 (Reuss et al.(2003) Cell Tissue Res. 313:139-157).

Fibroblast growth factor 21 (FGF21) was isolated from mouse embryos andis closest to FGF19 and FGF23. This FGF subfamily regulates diversephysiological processes uncommon to classical FGFs, namely energy andbile acid homeostasis, glucose and lipid metabolism, and phosphate aswell as vitamin D homeostasis. Moreover, unlike classical FGFs, thissubfamily acts in an endocrine fashion (Moore, D. D. (2007) Science 316,1436-8). FGF21 has been reported to be preferentially expressed in theliver (Nishimura et al. (2000) Biochimica et Biophysica Acta,1492:203-206; patent publication WO01/36640; and patent publicationWO01/18172) and described as a treatment for ischemic vascular disease,wound healing, and diseases associated with loss of pulmonary, bronchiaor alveolar cell function and numerous other disorders.

FGF21 has been identified as a potent metabolic regulator. Systemicadministration of FGF21 to rodents and rhesus monkeys with diet-inducedor genetic obesity and diabetes exerts strong anti-hyperglycemic andtriglyceride-lowering effects, and reduction of body weight (Coskun, T,et al. (2008) Endocrinology 149:6018-6027; Kharitonenkov, A. et al.(2005) Journal of Clinical Investigation 115:1627-1635; Kharitonenkov,A., et al. (2007) Endocrinology 148:774-781; Xu, J, et al. (2009)Diabetes 58:250-259). FGF21 is a 209 amino acid polypeptide containing a28 amino acid leader sequence. Human FGF21 has about 79% amino acididentity to mouse FGF21 and about 80% amino acid identity to rat FGF21.

In mammals, FGFs mediate their action via a set of four FGF receptorsFGFR1-4 that in turn are expressed in multiple spliced variants. EachFGF receptor contains an intracellular tyrosine kinase domain that isactivated upon ligand binding, leading to downstream signaling pathwaysinvolving MAPKs (Erk1/2), RAF1, AKT1 and STATs. (Kharitonenkov, A. etal. (2008) BioDrugs 22:37-44). Several reports suggested that the“c”-reporter splice variants of FGFR1-3 exhibit specific affinity toβ-klotho and could act as endogenous receptors for FGF21 (Kurosu et al.,2007 J. Biol. Chem. 282:26687-26695); Ogawa et al., 2007 Proc. Natl.Acad. Sci. USA 104:7432-7437; Kharitonenkov et al., 2008 J. CellPhysiol. 215, 1-7). In 3T3-L1 cells and white adipose tissue, FGFR1 isby far the most abundant receptor, and it is therefore most likely thatFGF21's main functional receptors in this tissue are the β-klotho-FGFR1ccomplexes.

Although FGF21 activates FGF receptors and downstream signalingmolecules, including FRS2a and ERK, direct interaction of FGFRs andFGF21 has not been detected. Furthermore, various non-adipocyte cells donot respond to FGF21, even though they express multiple FGFR isoforms.All of these data suggest that a cofactor must mediate FGF21 signalingthrough FGFRs. Studies have identified beta-klotho (β-klotho), which ishighly expressed in liver, adipocytes and in pancreas, as a determinantof the cellular response to FGF21 (Kurosu, H. et al. (2007) J Biol Chem282, 26687-95). The β-klotho-FGFR complex, but not FGFR alone, binds toFGF21 in vitro (Kharitonenkov, A., et al. (2008) J Cell Physiol 215,1-7). FGF21 binds to β-klotho in complex with FGFR1c, 2c, or 3c; but notto β-klotho in complex with FGFR4 (Owen et al., 2015 Trends inEndocrinology 26: 22-29). A similar mechanism has been identified in theFGF23-klotho-FGFR system (Urakawa, I. et al. (2006) Nature 444, 770-4).

The bioactivity of FGF21 was first identified in a mouse 3T3-L1adipocyte glucose uptake assay (Kharitonenkov, A. et al. (2005) J ClinInvest 115, 1627-35). Subsequently, FGF21 was shown to induceinsulin-independent glucose uptake and GLUT1 expression. FGF21 has alsobeen shown to ameliorate hyperglycemia in a range of diabetic rodentmodels. In addition, transgenic mice over-expressing FGF21 were found tobe resistant to diet-induced metabolic abnormalities, includingdecreased body weight and fat mass, and enhancements in insulinsensitivity (Badman, M. K. et al. (2007) Cell Metab 5, 426-37).Administration of FGF21 to diabetic non-human primates (NHP) caused adecline in fasting plasma glucose, triglycerides, insulin and glucagonlevels, and led to significant improvements in lipoprotein profilesincluding a nearly 80% increase in HDL cholesterol (Kharitonenkov, A. etal. (2007) Endocrinology 148, 774-81). Importantly, hypoglycemia was notobserved at any point during this NHP study. Other studies identifiedFGF21 as an important endocrine hormone that helps to control adaptationto the fasting state. This provides a previously missing link,downstream of PPARα, by which the liver communicates with the rest ofthe body in regulating the biology of energy homeostasis. The combinedobservations that FGF21 regulates adipose (lipolysis), liver (fatty acidoxidation and ketogenesis), and brain (torpor) establish it as a majorendocrine regulator of the response to fasting (Kharitonenkov, A. &Shanafelt, A. B. (2008) BioDrugs 22, 37-44).

The problem with using FGF21 directly as a biotherapeutic is that itshalf-life is very short. In mice, the half-life of human FGF21 is 0.5 to1 hours, and in cynomolgus monkeys, the half-life is 2 to 3 hours.Furthermore, when wild type FGF21 is used in pharmaceutical formulationsor preparations, its stability is adversely affected by preservativese.g., m-cresol.

SUMMARY

The present invention relates to FGF21 mimetic antibodies, i.e.,monoclonal antibodies that bind to beta-klotho (β-klotho) and activatethe human Fibroblast Growth Factor 21 (hereinafter, sometimes referredto as “FGF21”) receptor complex and FGF21-mediated signaling (e.g.,FGF21-receptor-dependent signaling), antigen-binding fragments thereof,and pharmaceutical compositions and methods of treatment comprising thesame.

Antigen-binding fragments (of the FGF21 mimetic, β-klotho-bindingantibodies) of the invention can be molecules with FGF21-like activityand selectivity but with added therapeutically desirable characteristicssuch as protein stability, low immunogenicity, ease of production and adesirable in vivo half-life.

The monoclonal FGF21 mimetic antibodies of the invention,antigen-binding fragments thereof, and pharmaceutical compositionscomprising the same are useful for the treatment of FGF21-associateddisorders, such as obesity, type 2 diabetes mellitus, type 1 diabetesmellitus, pancreatitis, dyslipidemia, nonalcoholic steatohepatitis(NASH), insulin resistance, hyperinsulinemia, glucose intolerance,hyperglycemia, metabolic syndrome, hypertension, cardiovascular disease,atherosclerosis, peripheral arterial disease, stroke, heart failure,coronary heart disease, kidney disease, diabetic complications,neuropathy, gastroparesis and other metabolic disorders, and in reducingthe mortality and morbidity of critically ill patients.

The isolated FGF21 mimetic antibodies, or antigen-binding fragments,described herein bind β-klotho, with an equilibrium dissociationconstant (K_(D)) of less than or equal to 100 pM. For example, theisolated antibodies or antigen-binding fragments described herein maybind to human β-klotho with a K_(D) of less than or equal to 100 pM,less than or equal to 50 pM, less than or equal to 45 pM, less than orequal to 40 pM, less than or equal to 35 pM, less than or equal to 25pM, or less than or equal to 15 pM. More specifically, the isolatedantibodies or antigen-binding fragments described herein may also bindhuman β-klotho with a K_(D) of less than or equal to 10 pM, as measuredby solution equilibrium titration assay (SET); and may also activate thecynomolgus monkey FGFR1c_β-klotho receptor complex with an EC50 of lessthan or equal to 50 nM, as measured by pERK cell assays.

The present invention relates to an isolated antibody, orantigen-binding fragments thereof, that binds to human and cynomolgusmonkey β-klotho. The invention also relates to an isolated antibody, orantigen-binding fragments thereof, that binds β-klotho and activates theFGF21 receptor complex and FGF21-mediated signaling (e.g.,FGF21-receptor-dependent signaling). In particular aspects, an isolatedantibody or antigen-binding fragment thereof described herein does notactivate human FGFR2c_β-klotho, FGFR3c_β-klotho, or FGFR4_0-klothoreceptor complexes.

The present invention also relates to an isolated antibody, orantigen-binding fragments thereof, that binds β-klotho and furthercompetes for binding with an antibody as described in Table 1. Thepresent invention also further relates to an isolated antibody, orantigen-binding fragments thereof, that binds the same epitope as anantibody as described in Table 1.

As described here, “competition” between antibodies and/orantigen-binding fragments thereof signifies that both antibodies (orbinding fragments thereof) bind to the same β-klotho epitope (e.g., asdetermined by a competitive binding assay, by any of the methods wellknown to those of skill in the art). An antibody or antigen-bindingfragment thereof also “competes” with a β-klotho antibody orantigen-binding fragment of the invention (e.g., NOV001 or NOV002) ifsaid competing antibody or antigen-binding fragment thereof binds thesame β-klotho epitope, or an overlapping β-klotho epitope, as anantibody or antigen-binding fragment of the invention. As used herein, acompeting antibody or antigen-binding fragment thereof can also includeone which (i) sterically blocks an antibody or antigen-binding fragmentof the invention from binding its target (e.g., if said competingantibody binds to a nearby, non-overlapping β-klotho and/or β-klothoepitope and physically prevents an antibody or antigen-binding fragmentof the invention from binding its target); and/or (ii) binds to adifferent, non-overlapping β-klotho epitope and induces a conformationalchange to the β-klotho protein such that said protein can no longer bebound by a β-klotho antibody or antigen-binding fragment of theinvention in a way that would occur absent said conformational change.

The binding affinity of isolated antibodies and antigen-bindingfragments described herein can be determined by solution equilibriumtitration (SET). Methods for SET are known in the art and are describedin further detail below. Alternatively, binding affinity of the isolatedantibodies, or fragments, described herein can be determined by Biacoreassay. Methods for Biacore kinetic assays are know in the art and aredescribed in further detail below.

The isolated FGF21 mimetic antibodies, or antigen-binding fragmentsthereof, may be used to increase the activation of the FGF21 receptorcomplex, and thereby, the FGF21 signaling pathway.

The isolated FGF21 mimetic antibodies, or antigen-binding fragmentsthereof, as described herein can be monoclonal antibodies, human orhumanized antibodies, chimeric antibodies, single chain antibodies, Fabfragments, Fv fragments, F(ab′)2 fragments, or scFv fragments, and/orIgG isotypes.

The isolated FGF21 mimetic antibodies, or antigen-binding fragmentsthereof, as described herein can also include a framework in which anamino acid has been substituted into the antibody framework from therespective human VH or VL germline sequences.

Another aspect of the invention includes an isolated antibody orantigen-binding fragments thereof having the full heavy and light chainsequences of Fabs described in Table 1. More specifically, the isolatedantibody or antigen-binding fragments thereof can have the heavy andlight chain sequences of Fab NOV001, NOV002, NOV003, NOV004.

A further aspect of the invention includes an isolated antibody orantigen-binding fragments thereof having the heavy and light chainvariable domain sequences of Fabs described in Table 1. Morespecifically, the isolated antibody or antigen-binding fragment thereofcan have the heavy and light chain variable domain sequence of FabNOV001, NOV002, NOV003, NOV004.

The invention also relates to an isolated antibody or antigen-bindingfragments thereof that includes a heavy chain CDR1 selected from thegroup consisting of SEQ ID NOs: 3, 23, 43, and 63; a heavy chain CDR2selected from the group consisting of SEQ ID NOs: 4, 24, 44, and 64; anda heavy chain CDR3 selected from the group consisting of SEQ ID NOs: 5,25, 45, and 65, wherein the isolated antibody or antigen-bindingfragments thereof binds to human β-klotho. In another aspect, suchisolated antibody or antigen-binding fragments thereof further includesa light chain CDR1 selected from the group consisting of SEQ ID NOs: 13,33, 53, and 73; a light chain CDR2 selected from the group consisting ofSEQ ID NOs: 14, 34, 54, and 74; and a light chain CDR3 selected from thegroup consisting of SEQ ID NOs: 15, 35, 55, and 75.

The invention also relates to an isolated antibody or antigen-bindingfragments thereof that includes a light chain CDR1 selected from thegroup consisting of SEQ ID NOs: 13, 33, 53, and 73; a light chain CDR2selected from the group consisting of SEQ ID NOs: 14, 34, 54, and 74;and a light chain CDR3 selected from the group consisting of SEQ ID NOs:15, 35, 55, and 75, wherein the isolated antibody or antigen-bindingfragments thereof binds to human β-klotho.

The invention also relates to an isolated antibody or antigen-bindingfragments thereof that binds β-klotho having HCDR1, HCDR2, and HCDR3 andLCDR1, LCDR2, and LCDR3, as defined by Kabat, wherein HCDR1, HCDR2, andHCDR3 comprises SEQ ID NOs: 3, 4, and 5, and LCDR1, LCDR2, LCDR3comprises SEQ ID NOs: 13, 14, and 15; or HCDR1, HCDR2, and HCDR3comprises SEQ ID NOs: 23, 24, and 25, and LCDR1, LCDR2, LCDR3 comprisesSEQ ID NOs: 33, 34, and 35; or HCDR1, HCDR2, and HCDR3 comprises SEQ IDNOs: 43, 44, and 45, and LCDR1, LCDR2, LCDR3 comprises SEQ ID NOs: 53,54, and 55; or HCDR1, HCDR2, and HCDR3 comprises SEQ ID NOs: 63, 64, and65, and LCDR1, LCDR2, LCDR3 comprises SEQ ID NOs: 73, 74, and 75.

The invention also relates to an isolated antibody or antigen-bindingfragments thereof that binds β-klotho having HCDR1, HCDR2, and HCDR3 andLCDR1, LCDR2, and LCDR3, as defined by Chothia, wherein HCDR1, HCDR2,and HCDR3 comprises SEQ ID NOs: 6, 7, and 8, and LCDR1, LCDR2, LCDR3comprises SEQ ID NOs: 16, 17, and 18; or HCDR1, HCDR2, and HCDR3comprises SEQ ID NOs: 26, 27, and 28, and LCDR1, LCDR2, LCDR3 comprisesSEQ ID NOs: 36, 37, and 38; or HCDR1, HCDR2, and HCDR3 comprises SEQ IDNOs: 46, 47, and 48, and LCDR1, LCDR2, LCDR3 comprises SEQ ID NOs: 56,57, and 58; or HCDR1, HCDR2, and HCDR3 comprises SEQ ID NOs: 66, 67, and68, and LCDR1, LCDR2, LCDR3 comprises SEQ ID NOs: 76, 77, and 78.

In one aspect of the invention the isolated antibody or antigen-bindingfragments thereof includes a heavy chain variable domain sequenceselected from the group consisting of SEQ ID NOs: 9, 29, 49, and 69. Theisolated antibody or antigen-binding fragment further can comprise alight chain variable domain sequence wherein the heavy chain variabledomain and light chain variable domain combine to form andantigen-binding site for β-klotho. In particular the light chainvariable domain sequence can be selected from SEQ ID NOs: 19, 39, 59,and 79 wherein said isolated antibody or antigen-binding fragmentsthereof binds beta-klotho.

The invention also relates to an isolated antibody or antigen-bindingfragments thereof that includes a light chain variable domain sequenceselected from the group consisting of SEQ ID NOs: 19, 39, 59, and 79,wherein said isolated antibody or antigen-binding fragments thereofbinds to human β-klotho. The isolated antibody or antigen-bindingfragment may further comprise a heavy chain variable domain sequencewherein the light chain variable domain and heavy chain variable domaincombine to form and antigen-binding site for β-klotho.

In particular, the isolated antibody or antigen-binding fragmentsthereof that binds β-klotho, may have heavy and light chain variabledomains comprising the sequences of SEQ ID NOs: 9 and 19; 29 and 39; 49and 59; or 69 and 79, respectively.

The invention further relates to an isolated antibody or antigen-bindingfragments thereof, that includes a heavy chain variable domain having atleast 90% sequence identity to a sequence selected from the groupconsisting of SEQ ID NOs: 9, 29, 49, and 69, wherein said antibody bindsto β-klotho. In one aspect, the isolated antibody or antigen-bindingfragments thereof also includes a light chain variable domain having atleast 90% sequence identity to a sequence selected from the groupconsisting of SEQ ID NOs: 19, 39, 59, and 79. In a further aspect of theinvention, the isolated antibody or antigen-binding fragment has anHCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as defined by Kabat and asdescribed in Table 1.

The invention also relates to an isolated antibody or antigen-bindingfragments thereof, having a light chain variable domain having at least90% sequence identity to a sequence selected from the group consistingof SEQ ID NOs: 19, 39, 59, and 79, wherein said antibody binds β-klotho.

In another aspect of the invention, the isolated antibody, orantigen-binding fragments thereof, that binds to β-klotho may have aheavy chain comprising the sequence of SEQ ID NOs: 11, 31, 51, or 71.The isolated antibody can also includes a light chain that can combinewith the heavy chain to form an antigen-binding site to human β-klotho.In particular, the light chain may have a sequence comprising SEQ IDNOs: 21, 41, 61, or 81. In particular, the isolated antibody orantigen-binding fragments thereof that binds β-klotho, may have a heavychain and a light chain comprising the sequences of SEQ ID NOs: 11 and21; 31 and 41; 51 and 61; or 71 and 81, respectively.

The invention still further relates to an isolated antibody orantigen-binding fragments thereof that includes a heavy chain having atleast 90% sequence identity to a sequence selected from the groupconsisting of SEQ ID NOs: 9, 29, 49, or 69, wherein said antibody bindsto β-klotho. In one aspect, the isolated antibody or antigen-bindingfragments thereof also includes a light chain having at least 90%sequence identity to a sequence selected from the group consisting ofSEQ ID NOs: 21, 41, 61, or 81.

The invention still further relates to an isolated antibody orantigen-binding fragments thereof that includes a light chain having atleast 90% sequence identity to a sequence selected from the groupconsisting of SEQ ID NOs: 21, 41, 61, or 81, wherein said antibody binds3-klotho.

The invention also relates to compositions comprising the isolatedantibody, or antigen-binding fragments thereof, described herein. Aswell as, antibody compositions in combination with a pharmaceuticallyacceptable carrier. Specifically, the invention further includespharmaceutical compositions comprising an antibody or antigen-bindingfragments thereof of Table 1, such as, for example antibody NOV001,NOV002, NOV003, NOV004. The invention also relates to pharmaceuticalcompositions comprising a combination of two or more of the isolatedantibodies or antigen-binding fragments thereof of Table 1.

The invention also relates to an isolated nucleic acid sequence encodingthe variable heavy chain having a sequence selected from SEQ ID NOs: 9,29, 49, and 69. In particular the nucleic acid has a sequence at least90% sequence identity to a sequence selected from the group consistingof SEQ ID NOs: 10, 30, 50, and 70. In a further aspect of the inventionthe sequence is SEQ ID NOs: 10, 30, 50, and 70.

The invention also relates to an isolated nucleic acid sequence encodingthe variable light chain having a sequence selected from SEQ ID NOs: 20,40, 60, and 80. In particular the nucleic acid has a sequence at least90% sequence identity to a sequence selected from the group consistingof SEQ ID NOs: 20, 40, 60, and 80. In a further aspect of the inventionthe sequence is SEQ ID NOs: 20, 40, 60, and 80.

The invention also relates to an isolated nucleic acid comprising asequence encoding a polypeptide that includes a light chain variabledomain having at least 90% sequence identity to a sequence selected fromthe group consisting of SEQ ID NOs: 20, 40, 60, and 80.

The invention also relates to a vector that includes one or more of thenucleic acid molecules described herein.

The invention also relates to an isolated host cell that includes arecombinant DNA sequence encoding a heavy chain of the antibodydescribed above, and a second recombinant DNA sequence encoding a lightchain of the antibody described above, wherein said DNA sequences areoperably linked to a promoter and are capable of being expressed in thehost cell. It is contemplated that the antibody can be a humanmonoclonal antibody. It is also contemplated that the host cell is anon-human mammalian cell.

The invention also relates to activating a Fibroblast Growth Factor 21(FGF21) receptor, and, thereby, FGF21-mediated signaling (e.g.,FGF21-receptor-dependent signaling), wherein the method includes thestep of contacting a cell with an effective amount of a compositioncomprising the isolated antibody or antigen-binding fragments thereofdescribed herein.

It is contemplated that the cell is a human cell. It is furthercontemplated that the cell is in a subject. In one embodiment, it iscontemplated that the cell is an adipocyte. In other embodiments, thecell may be one or more of hepatocytes, pancreas cells, endothelialcells, muscle, or renal cells. It is still further contemplated that thesubject is human.

The invention also relates to a method of treating, improving, orpreventing a FGF21-associated disorder in a subject, wherein the methodincludes the step of administering to the subject an effective amount ofa composition comprising the antibody or antigen-binding fragmentsthereof described herein. In one aspect, the FGF21-associated disorderis obesity. In one aspect, the FGF21-associated disorder is type 2diabetes. It is contemplated that the subject is human.

Any of the foregoing isolated antibodies or antigen-binding fragmentsthereof may be a monoclonal antibody or antigen-binding fragmentsthereof.

Non-limiting embodiments of the disclosure are described in thefollowing aspects:

1. An isolated antibody or antigen-binding fragment thereof that bindsto an epitope within the extracellular domain of β-klotho.

2. An isolated antibody or antigen-binding fragment thereof that bindsto an epitope of β-klotho, wherein the epitope comprises one or more ofthe SEQ ID NOs shown in Table 2.

3. An isolated FGF21 mimetic antibody or antigen-binding fragmentthereof that binds to β-klotho, wherein said antibody or fragmentincreases the activity of β-klotho and FGFR1c.

4. An isolated antibody or antigen-binding fragment thereof that bindsto a human β-klotho protein with a K_(D) of less than or equal to 10 pM,as measured by solution equilibrium titration assay (SET).

5. An isolated antibody or antigen-binding fragment thereof that bindsto an epitope of β-klotho, wherein said epitope comprises one or moreamino acids of residues 246-265, 536-550, 834-857 and 959-986 of theβ-klotho sequence (SEQ ID NO:262).

6. An isolated antibody or antigen-binding fragment thereof that bindsto one or more epitopes of β-klotho, wherein said epitopes comprises oneor more of amino acids of residues 646-670, 696-700, and 646-689 of theβ-klotho sequence (SEQ ID NO:262).

7. An isolated antibody or antigen-binding fragment thereof thatactivates the cynomolgus monkey FGFR1c_β-klotho receptor complex with anEC50 of less than or equal to 50 nM, as measured by pERK cell assays.

8. The isolated antibody or antigen-binding fragment of aspect 1,wherein said antibody or fragment comprises at least one complementaritydetermining region having at least 95% identity to at least one of theCDRs recited in Table 1.

9. The isolated antibody or antigen-binding fragment of aspect 1,wherein said antibody or fragment comprises at least one complementaritydetermining region having at least 98% identity to at least one of theCDRs recited in Table 1.

10. The isolated antibody or antigen-binding fragment of aspect 1,wherein said antibody or fragment comprises at least one complementaritydetermining region having at least 99% identity to at least one of theCDRs recited in Table 1.

11. The isolated antibody or antigen-binding fragment of any of thepreceding aspects, wherein said antibody or fragment comprises a heavychain CDR1, heavy chain CDR2, and heavy chain CDR3 from Table 1, and/ora light chain CDR1, light chain CDR2, and light chain CDR3 from Table 1.

12. The isolated antibody or antigen-binding fragment of any of aspects1-7, wherein said antibody or fragment comprises a CDR1, CDR2, and CDR3from Table 1, and wherein the variant has at least one to four aminoacid changes in one of CDR1, CDR2, or CDR3.

13. The isolated antibody or antigen-binding fragment of any of aspects1-7, wherein the antibody or fragment comprises a heavy chain CDR3selected from the group consisting of SEQ ID NO: 5, 25, 45, and 65.

14. The isolated antibody or antigen-binding fragment of any of aspects1-7, wherein the antibody or fragment comprises a VH selected from thegroup consisting of SEQ ID NO: 9, 29, 49, 69 or an amino acid sequencewith 90% identity thereof; and a VL selected from the group consistingof SEQ ID NO: 19, 39, 59, and 79 or an amino acid sequence with 90%identity thereof.

15. The isolated antibody or antigen-binding fragment of any of aspects1-7, wherein the antibody or fragment comprises a VH selected from thegroup consisting of SEQ ID NO: 9, 29, 49, and 29 or an amino acidsequence with 95% identity thereof; and a VL selected from the groupconsisting of SEQ ID NO: 19, 39, 59, and 79 or an amino acid sequencewith 95% identity thereof.

16. The isolated antibody or antigen-binding fragment of any of aspects1-7, wherein the antibody or fragment comprises a VH selected from thegroup consisting of SEQ ID NO: 9, 29, 49, and 69 or an amino acidsequence with 97% identity thereof, and a VL selected from the groupconsisting of SEQ ID NO: 19, 39, 59, and 79 or an amino acid sequencewith 97% identity thereof.

17. The isolated antibody or antigen-binding fragment of any of aspects1-7, wherein the antibody or fragment comprises a variable heavy chainsequence selected from the group consisting of SEQ ID NO: 9, 29, 49, and69.

18. The isolated antibody or antigen-binding fragment of any of aspects1-7, wherein the antibody or fragment comprises a variable light chainsequence selected from the group consisting of SEQ ID NO: 19, 39, 59,and 79.

19. The isolated antibody or antigen-binding fragment of any of aspects1-7, wherein the antibody or fragment comprises a variable heavy chainselected from the group consisting of SEQ ID NO: 9, 29, 49, and 69; andvariable light chain sequence selected from the group consisting of SEQID NO: 19, 39, 59, and 79.

20. The isolated antibody or antigen-binding fragment of any of aspects1-7, wherein the antibody or fragment is selected from the groupconsisting of an antibody or fragment comprising a variable heavy chainof SEQ ID NO: 9 and a variable light chain sequence of SEQ ID NO: 19, anantibody or fragment comprising a variable heavy chain of SEQ ID NO: 29and a variable light chain sequence of SEQ ID NO: 39; an antibody orfragment comprising a variable heavy chain of SEQ ID NO: 49 and avariable light chain sequence of SEQ ID NO: 59; and an antibody orfragment comprising a variable heavy chain of SEQ ID NO: 69 and avariable light chain sequence of SEQ ID NO: 79.

21. The isolated antibody or antigen-binding fragment of any of aspects1-7, wherein the antibody or fragment comprises a heavy chain CDR1selected from the group consisting of SEQ ID NO: 3, 23, 43, and 63; aheavy chain CDR2 selected from the group consisting of SEQ ID NO: 4, 24,44, and 64; a heavy chain CDR3 selected from the group consisting of 5,25, 45, and 65; a light chain CDR1 selected from the group consisting ofSEQ ID NO: 13, 33, 53, and 73; a light chain CDR2 selected from thegroup consisting of SEQ ID NO: 14, 34, 54, and 74; and a light chainCDR3 selected from the group consisting of SEQ ID NO: 15, 35, 55, and75.

22. The isolated antibody or antigen-binding fragment of any of aspects1-7, wherein the antibody or fragment comprises a heavy chain CDR1selected from the group consisting of SEQ ID NO: 6, 26, 46, and 66; aheavy chain CDR2 selected from the group consisting of SEQ ID NO: 7, 27,47, and 67; a heavy chain CDR3 selected from the group consisting of 8,28, 48, and 68; a light chain CDR1 selected from the group consisting ofSEQ ID NO: 16, 36, 56, and 76; a light chain CDR2 selected from thegroup consisting of SEQ ID NO: 17, 37, 57, and 77; and a light chainCDR3 selected from the group consisting of SEQ ID NO: 18, 38, 58, and78.

23. The isolated antibody or antigen-binding fragment of any of aspects1-7, wherein the antibody or fragment comprises a heavy chain CDR1 ofSEQ ID NO: 3; a heavy chain CDR2 of SEQ ID NO: 4; a heavy chain CDR3 ofSEQ ID NO: 5; a light chain CDR1 of SEQ ID NO: 13; a light chain CDR2 ofSEQ ID NO: 14; and a light chain CDR3 of SEQ ID NO: 15.

24. The isolated antibody or antigen-binding fragment of any of aspects1-7, wherein the antibody or fragment comprises a heavy chain CDR1 ofSEQ ID NO: 23; a heavy chain CDR2 of SEQ ID NO: 24; a heavy chain CDR3of SEQ ID NO: 25; a light chain CDR1 of SEQ ID NO: 33; a light chainCDR2 of SEQ ID NO: 34; and a light chain CDR3 of SEQ ID NO: 35.

25. The isolated antibody or antigen-binding fragment of any of aspects1-7, wherein the antibody or fragment comprises a heavy chain CDR1 ofSEQ ID NO: 43; a heavy chain CDR2 of SEQ ID NO: 44; a heavy chain CDR3of SEQ ID NO: 45; a light chain CDR1 of SEQ ID NO: 53; a light chainCDR2 of SEQ ID NO: 54; and a light chain CDR3 of SEQ ID NO: 55.

26. The isolated antibody or antigen-binding fragment of any of aspects1-7, wherein the antibody or fragment comprises a heavy chain CDR1 ofSEQ ID NO: 63; a heavy chain CDR2 of SEQ ID NO: 64; a heavy chain CDR3of SEQ ID NO: 65; a light chain CDR1 of SEQ ID NO: 73; a light chainCDR2 of SEQ ID NO: 74; and a light chain CDR3 of SEQ ID NO: 75.

27. The isolated antibody or antigen-binding fragment of any of aspects1-7, wherein the antibody or fragment comprises a heavy chain CDR1 ofSEQ ID NO: 6; a heavy chain CDR2 of SEQ ID NO: 7; a heavy chain CDR3 ofSEQ ID NO: 8; a light chain CDR1 of SEQ ID NO: 16; a light chain CDR2 ofSEQ ID NO: 17; and a light chain CDR3 of SEQ ID NO: 18.

28. The isolated antibody or antigen-binding fragment of any of aspects1-7, wherein the antibody or fragment comprises a heavy chain CDR1 ofSEQ ID NO: 26; a heavy chain CDR2 of SEQ ID NO: 27; a heavy chain CDR3of SEQ ID NO: 28; a light chain CDR1 of SEQ ID NO: 36; a light chainCDR2 of SEQ ID NO: 37; and a light chain CDR3 of SEQ ID NO: 38.

29. The isolated antibody or antigen-binding fragment of any of aspects1-7, wherein the antibody or fragment comprises a heavy chain CDR1 ofSEQ ID NO: 46; a heavy chain CDR2 of SEQ ID NO: 47; a heavy chain CDR3of SEQ ID NO: 48; a light chain CDR1 of SEQ ID NO: 56; a light chainCDR2 of SEQ ID NO: 57; and a light chain CDR3 of SEQ ID NO: 58.

30. The isolated antibody or antigen-binding fragment of any of aspects1-7, wherein the antibody or fragment comprises a heavy chain CDR1 ofSEQ ID NO: 66; a heavy chain CDR2 of SEQ ID NO: 67; a heavy chain CDR3of SEQ ID NO: 68; a light chain CDR1 of SEQ ID NO: 76; a light chainCDR2 of SEQ ID NO: 77; and a light chain CDR3 of SEQ ID NO: 78.

31. An isolated antibody or antigen-binding fragment thereof, whereinthe antibody or fragment binds to the same epitope as an isolatedantibody or fragment according to any of aspects 12-30.

32. An isolated antibody or antigen-binding fragment thereof, whereinthe antibody or fragment competes for binding to β-klotho with anisolated antibody or fragment according to any of aspects 12-30.

33. The isolated antibody or antigen-binding fragment of any of aspects1-7, wherein the antibody or fragment is selected from the groupconsisting of NOV001, NOV002, NOV003, and NOV004.

34. A pharmaceutical composition comprising an antibody orantigen-binding fragment thereof of one of the above aspects and apharmaceutically acceptable carrier.

35. A method of treating a metabolic disorder comprising administeringto a subject afflicted with a metabolic disorder an effective amount ofa pharmaceutical composition comprising an antibody or antigen-bindingfragment according to any of aspects 1-30.

36. The method of aspect 35, wherein the subject is afflicted with oneor more of obesity, type 1 and type 2 diabetes mellitus, pancreatitis,dyslipidemia, nonalcoholic steatohepatitis (NASH), insulin resistance,hyperinsulinemia, glucose intolerance, hyperglycemia, and metabolicsyndrome.

37. The method of aspect 35, wherein the subject is afflicted with oneor more of obesity, diabetes, and dyslipidemia.

38. A method of treating a cardiovascular disorder comprisingadministering to a subject afflicted with a cardiovascular disorder aneffective amount of a pharmaceutical composition comprising an antibodyor fragment according to any previous aspect.

39. The method of aspect 38, wherein the subject is afflicted with oneor more of atherosclerosis, peripheral arterial disease, stroke, heartfailure, and coronary heart disease.

40. An antibody or antigen-binding fragment thereof according to any ofaspects 1-30, for use as a medicament.

41. A nucleic acid coding for one or more of the antibodies according toany previous aspect.

42. A nucleic acid comprising a sequence with at least 90% identity tothe sequences set forth in Table 1.

43. A nucleic acid comprising a sequence with at least 95% identity tothe sequences set forth in Table 1.

44. A vector comprising the nucleic acid according to aspect 41.

45. A host cell comprising the vector of aspect 44.

46. A pharmaceutical composition comprising an antibody orantigen-binding fragment according to any of aspects 1-30 for use intreating a metabolic disorder.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which this invention pertains.

As used herein, the term “FGF21” refers to a member of the fibroblastgrowth factor (FGF) protein family. An amino acid sequence of FGF21(GenBank Accession No. NP_061986.1) is set forth as SEQ ID NO:1, thecorresponding polynucleotide sequence of which is set forth as SEQ IDNO:2 (NCBI reference sequence number NM_019113.2).

As used herein, the term “FGF21 receptor” refers to a receptor for FGF21(Kharitonenkov, A, et al. (2008) Journal of Cellular Physiology 215:1-7;Kurosu, H, et al. (2007) JBC 282:26687-26695; Ogawa, Y, et al. (2007)PNAS 104:7432-7437).

The term “FGF21 polypeptide” refers to a naturally-occurring polypeptideexpressed in humans. For purposes of this disclosure, the term “FGF21polypeptide” can be used interchangeably to refer to any full-lengthFGF21 polypeptide, e.g., SEQ ID NO:1, which consists of 209 amino acidresidues and which is encoded by the nucleotide sequence of SEQ ID NO:2;any mature form of the polypeptide, which consists of 181 amino acidresidues, and in which the 28 amino acid residues at the amino-terminalend of the full-length FGF21 polypeptide (i.e., which constitute thesignal peptide) have been removed, and variants thereof.

The term “antibody” as used herein means a whole antibody and anyantigen-binding fragment (i.e., “antigen-binding portion”) or singlechain thereof. A whole antibody is a glycoprotein comprising at leasttwo heavy (H) chains and two light (L) chains inter-connected bydisulfide bonds. Each heavy chain is comprised of a heavy chain variableregion (abbreviated herein as VH) and a heavy chain constant region. Theheavy chain constant region is comprised of three domains, CH1, CH2 andCH3. Each light chain is comprised of a light chain variable region(abbreviated herein as VL) and a light chain constant region. The lightchain constant region is comprised of one domain, CL. The VH and VLregions can be further subdivided into regions of hypervariability,termed complementarity determining regions (CDR), interspersed withregions that are more conserved, termed framework regions (FR). Each VHand VL is composed of three CDRs and four FRs arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and lightchains contain a binding domain that interacts with an antigen. Theconstant regions of the antibodies may mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (C1q)of the classical complement system.

The term “antigen-binding portion” or “antigen-binding fragment” of anantibody, as used herein, refers to one or more fragments of an intactantibody that retain the ability to specifically bind to a given antigen(e.g., β-klotho). Antigen-binding functions of an antibody can beperformed by fragments of an intact antibody. Examples of bindingfragments encompassed within the term antigen-binding portion orantigen-binding fragment of an antibody include a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; aF(ab)₂ fragment, a bivalent fragment comprising two Fab fragments linkedby a disulfide bridge at the hinge region; an Fd fragment consisting ofthe VH and CH1 domains; an Fv fragment consisting of the VL and VHdomains of a single arm of an antibody; a single domain antibody (dAb)fragment (Ward et al., 1989 Nature 341:544-546), which consists of a VHdomain or a VL domain; and an isolated complementarity determiningregion (CDR).

Furthermore, although the two domains of the Fv fragment, VL and VH, arecoded for by separate genes, they can be joined, using recombinantmethods, by an artificial peptide linker that enables them to be made asa single protein chain in which the VL and VH regions pair to formmonovalent molecules (known as single chain Fv (scFv); see, e.g., Birdet al., 1988 Science 242:423-426; and Huston et al., 1988 Proc. Natl.Acad. Sci. 85:5879-5883). Such single chain antibodies include one ormore antigen-binding portions or fragments of an antibody. Theseantibody fragments are obtained using conventional techniques known tothose of skill in the art, and the fragments are screened for utility inthe same manner as are intact antibodies.

Antigen-binding fragments can also be incorporated into single domainantibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies,tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, 2005,Nature Biotechnology, 23, 9, 1126-1136). Antigen-binding portions ofantibodies can be grafted into scaffolds based on polypeptides such asFibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describesfibronectin polypeptide monobodies).

Antigen-binding fragments can be incorporated into single chainmolecules comprising a pair of tandem Fv segments (VH-CH1-VH-CH1) which,together with complementary light chain polypeptides, form a pair ofantigen-binding regions (Zapata et al. (1995) Protein Eng.8(10):1057-1062; and U.S. Pat. No. 5,641,870).

As used herein, the term “affinity” refers to the strength ofinteraction between antibody and antigen at single antigenic sites.Within each antigenic site, the variable region of the antibody “arm”interacts through weak non-covalent forces with antigen at numeroussites; the more interactions, the stronger the affinity. As used herein,the term “high affinity” for an antibody or antigen-binding fragmentsthereof (e.g., a Fab fragment) generally refers to an antibody, orantigen-binding fragment, having a KD of 10⁻⁹M or less.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refer to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an alpha carbon that is boundto a hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

The term “binding specificity” as used herein refers to the ability ofan individual antibody combining site to react with only one antigenicdeterminant.

The phrase “specifically (or selectively) binds” to an antibody (e.g., aβ-klotho-binding antibody) refers to a binding reaction that isdeterminative of the presence of a cognate antigen (e.g., a humanβ-klotho or cynomolgus β-klotho) in a heterogeneous population ofproteins and other biologics. The phrases “an antibody recognizing anantigen” and “an antibody specific for an antigen” are usedinterchangeably herein with the term “an antibody which bindsspecifically to an antigen”.

The term “FGF21 mediated” or similar refers to the fact that the FGF21receptor and/or the antibodies of the invention mediate the cellularresponse and the FGF21 signaling pathway upon binding to β-klotho,thereby triggering a variety of physiological effects, including but notlimited to a reduction in one or more of the following: plasmatriglycerides, plasma insulin, plasma glucose, food intake, and bodyweight.

An “FGF21-associated disorder,” “FGF21-associated condition,” “diseaseor condition associated with FGF21,” or similar terms as used herein,refer to any number of conditions or diseases for which the prevention,diagnosis, and/or treatment by activation of the FGF21 signaling pathway(e.g., by activation of FGF21 receptor signaling), is sought. These caninclude conditions, diseases, or disorders characterized by aberrantFGF21 signaling (e.g., aberrant activation of FGF21-mediated signalingand/or FGF21 receptor signaling). These conditions include but are notlimited to metabolic, endocrine, and cardiovascular disorders, such asobesity, type 1 and type 2 diabetes mellitus, pancreatitis,dyslipidemia, nonalcoholic fatty liver disease (NAFLD), nonalcoholicsteatohepatitis (NASH), insulin resistance, hyperinsulinemia, glucoseintolerance, hyperglycemia, metabolic syndrome, acute myocardialinfarction, hypertension, cardiovascular disease, atherosclerosis,peripheral arterial disease, stroke, heart failure, coronary heartdisease, kidney disease, diabetic complications, neuropathy,gastroparesis, disorders associated with severe inactivating mutationsin the insulin receptor, and other metabolic disorders, and in reducingthe mortality and morbidity of critically ill patients.

“Type 2 diabetes mellitus” is a condition characterized by excessglucose production in spite of the availability of insulin, andcirculating glucose levels remain excessively high as a result ofinadequate glucose clearance.

“Type 1 diabetes mellitus” is a condition characterized by high bloodglucose levels caused by total lack of insulin. This occurs when thebody's immune system attacks the insulin-producing beta cells in thepancreas and destroys them. The pancreas then produces little or noinsulin.

“Pancreatitis” is inflammation of the pancreas.

“Dyslipidemia” is a disorder of lipoprotein metabolism, includinglipoprotein overproduction or deficiency. Dyslipidemias may bemanifested by elevation of the total cholesterol, low-densitylipoprotein (LDL) cholesterol and triglyceride concentrations, and adecrease in high-density lipoprotein (HDL) cholesterol concentration inthe blood.

“Nonalcoholic steatohepatitis (NASH)” is a liver disease, not associatedwith alcohol consumption, characterized by fatty change of hepatocytes,accompanied by intralobular inflammation and fibrosis.

“Glucose intolerance,” or Impaired Glucose Tolerance (IGT) is apre-diabetic state of dysglycemia that is associated with increased riskof cardiovascular pathology. The pre-diabetic condition prevents asubject from moving glucose into cells efficiently and utilizing it asan efficient fuel source, leading to elevated glucose levels in bloodand some degree of insulin resistance.

“Hyperglycemia” is defined as an excess of sugar (glucose) in the blood.

“Hypoglycemia”, also called low blood sugar, occurs when your bloodglucose level drops too low to provide enough energy for your body'sactivities.

“Hyperinsulinemia” is defined as a higher-than-normal level of insulinin the blood.

“Insulin resistance” is defined as a state in which a normal amount ofinsulin produces a subnormal biologic response.

“Obesity,” in terms of the human subject, can be defined as that bodyweight over 20 percent above the ideal body weight for a givenpopulation (R. H. Williams, Textbook of Endocrinology, 1974, p.904-916). It can also be defined as a Body Mass Index (BMI, defined as aperson's weight in kilograms divided by the square of his height inmeters (kg/m2)) as greater than or equal to 30.

“Metabolic syndrome” can be defined as a cluster of at least three ofthe following signs: abdominal fat—in most men, a 40-inch waist orgreater; high blood sugar—at least 110 milligrams per deciliter (mg/dl)after fasting; high triglycerides—at least 150 mg/dL in the bloodstream;low HDL—less than 40 mg/dl; and, blood pressure of 130/85 mmHg orhigher.

“Hypertension” or high blood pressure that is a transitory or sustainedelevation of systemic arterial blood pressure to a level likely toinduce cardiovascular damage or other adverse consequences. Hypertensionhas been arbitrarily defined as a systolic blood pressure above 140 mmHgor a diastolic blood pressure above 90 mmHg.

“Cardiovascular diseases” are diseases related to the heart or bloodvessels.

“Peripheral arterial disease” occurs when plaque builds up in thearteries that carry blood to the head, organs and limbs. Over time,plaque can harden and narrow the arteries which limits the flow ofoxygen-rich blood to organs and other parts of the body.

“Atherosclerosis” is a vascular disease characterized by irregularlydistributed lipid deposits in the intima of large and medium-sizedarteries, causing narrowing of arterial lumens and proceeding eventuallyto fibrosis and calcification. Lesions are usually focal and progressslowly and intermittently. Limitation of blood flow accounts for mostclinical manifestations, which vary with the distribution and severityof lesions.

“Stroke” is any acute clinical event, related to impairment of cerebralcirculation, that lasts longer than 24 hours. A stroke involvesirreversible brain damage, the type and severity of symptoms dependingon the location and extent of brain tissue whose circulation has beencompromised.

“Heart failure”, also called congestive heart failure, is a condition inwhich the heart can no longer pump enough blood to the rest of the body.

“Coronary heart disease”, also called coronary artery disease, is anarrowing of the small blood vessels that supply blood and oxygen to theheart.

“Kidney disease” or nephropathy is any disease of the kidney. Diabeticnephropathy is a major cause of morbidity and mortality in people withtype 1 or type 2 diabetes mellitus.

“Diabetic complications” are problems, caused by high blood glucoselevels, with other body functions such as kidneys, nerves(neuropathies), feet (foot ulcers and poor circulation) and eyes (e.g.retinopathies). Diabetes also increases the risk for heart disease andbone and joint disorders. Other long-term complications of diabetesinclude skin problems, digestive problems, sexual dysfunction andproblems with teeth and gums.

“Neuroapathies” are any diseases involving the cranial nerves or theperipheral or autonomic nervous system.

“Gastroparesis” is weakness of gastric peristalsis, which results indelayed emptying of the bowels.

The critically ill patients encompassed by the present inventiongenerally experience an unstable hypermetabolic state. This unstablemetabolic state is due to changes in substrate metabolism, which maylead to relative deficiencies in some nutrients. Generally there is anincreased oxidation of both fat and muscle.

Moreover, critically ill patients are preferably patients thatexperience systemic inflammatory response syndrome or respiratorydistress. A reduction in morbidity means reducing the likelihood that acritically ill patient will develop additional illnesses, conditions, orsymptoms or reducing the severity of additional illnesses, conditions,or symptoms. For example reducing morbidity may correspond to a decreasein the incidence of bacteremia or sepsis or complications associatedwith multiple organ failure.

The term “conservatively modified variant” applies to both amino acidand nucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidthat encodes a polypeptide is implicit in each described sequence.

For polypeptide sequences, “conservatively modified variants” includeindividual substitutions, deletions or additions to a polypeptidesequence which result in the substitution of an amino acid with achemically similar amino acid. Conservative substitution tablesproviding functionally similar amino acids are well known in the art.Such conservatively modified variants are in addition to and do notexclude polymorphic variants, interspecies homologs, and alleles of theinvention. The following eight groups contain amino acids that areconservative substitutions for one another: 1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine(Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L),Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M) (see, e.g., Creighton, Proteins (1984)). In someembodiments, the term “conservative sequence modifications” are used torefer to amino acid modifications that do not significantly affect oralter the binding characteristics of the antibody containing the aminoacid sequence.

The term “epitope” means a protein determinant capable of specificbinding to an antibody. Epitopes usually consist of chemically activesurface groupings of molecules such as amino acids or sugar side chainsand usually have specific three dimensional structural characteristics,as well as specific charge characteristics. Conformational andnonconformational epitopes are distinguished in that the binding to theformer but not the latter is lost in the presence of denaturingsolvents.

The term “human antibody”, as used herein, is intended to includeantibodies having variable regions in which both the framework and CDRregions are derived from sequences of human origin. Furthermore, if theantibody contains a constant region, the constant region also is derivedfrom such human sequences, e.g., human germline sequences, or mutatedversions of human germline sequences. The human antibodies of theinvention may include amino acid residues not encoded by human sequences(e.g., mutations introduced by random or site-specific mutagenesis invitro or by somatic mutation in vivo).

The term “human monoclonal antibody” refers to antibodies displaying asingle binding specificity which have variable regions in which both theframework and CDR regions are derived from human sequences. In oneembodiment, the human monoclonal antibodies are produced by a hybridomawhich includes a B cell obtained from a transgenic nonhuman animal,e.g., a transgenic mouse, having a genome comprising a human heavy chaintransgene and a light chain transgene fused to an immortalized cell.

A “humanized” antibody is an antibody that retains the reactivity of anon-human antibody while being less immunogenic in humans. This can beachieved, for instance, by retaining the non-human CDR regions andreplacing the remaining parts of the antibody with their humancounterparts (i.e., the constant region as well as the frameworkportions of the variable region). See, e.g., Morrison et al., Proc.Natl. Acad. Sci. USA, 81:6851-6855, 1984; Morrison and Oi, Adv.Immunol., 44:65-92, 1988; Verhoeyen et al., Science, 239:1534-1536,1988; Padlan, Molec. Immun., 28:489-498, 1991; and Padlan, Molec.Immun., 31:169-217, 1994. Other examples of human engineering technologyinclude, but are not limited to Xoma technology disclosed in U.S. Pat.No. 5,766,886.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same. Two sequences are“substantially identical” if two sequences have a specified percentageof amino acid residues or nucleotides that are the same (i.e., 60%identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identityover a specified region, or, when not specified, over the entiresequence), when compared and aligned for maximum correspondence over acomparison window, or designated region as measured using one of thefollowing sequence comparison algorithms or by manual alignment andvisual inspection. Optionally, the identity exists over a region that isat least about 50 nucleotides (or 10 amino acids) in length, or morepreferably over a region that is 100 to 500 or 1000 or more nucleotides(or 20, 50, 200 or more amino acids) in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homologyalignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443, 1970,by the search for similarity method of Pearson and Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444, 1988, by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., Brent etal., Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(Ringbou ed., 2003)).

Two examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al., Nuc. Acids Res.25:3389-3402, 1977; and Altschul et al., J. Mol. Biol. 215:403-410,1990, respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information.This algorithm involves first identifying high scoring sequence pairs(HSPs) by identifying short words of length W in the query sequence,which either match or satisfy some positive-valued threshold score Twhen aligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold (Altschul et al.,supra). These initial neighborhood word hits act as seeds for initiatingsearches to find longer HSPs containing them. The word hits are extendedin both directions along each sequence for as far as the cumulativealignment score can be increased. Cumulative scores are calculatedusing, for nucleotide sequences, the parameters M (reward score for apair of matching residues; always >0) and N (penalty score formismatching residues; always <0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLASTN program(for nucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) or 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989)alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul, Proc.Natl. Acad. Sci. USA 90:5873-5787, 1993). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

The percent identity between two amino acid sequences can also bedetermined using the algorithm of E. Meyers and W. Miller (Comput. Appl.Biosci., 4:11-17, 1988) which has been incorporated into the ALIGNprogram (version 2.0), using a PAM120 weight residue table, a gap lengthpenalty of 12 and a gap penalty of 4. In addition, the percent identitybetween two amino acid sequences can be determined using the Needlemanand Wunsch (J. Mol, Biol. 48:444-453, 1970) algorithm which has beenincorporated into the GAP program in the GCG software package (availableon the world wide web at gcg.com), using either a Blossom 62 matrix or aPAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and alength weight of 1, 2, 3, 4, 5, or 6.

Other than percentage of sequence identity noted above, anotherindication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid, asdescribed below. Thus, a polypeptide is typically substantiallyidentical to a second polypeptide, for example, where the two peptidesdiffer only by conservative substitutions. Another indication that twonucleic acid sequences are substantially identical is that the twomolecules or their complements hybridize to each other under stringentconditions, as described below. Yet another indication that two nucleicacid sequences are substantially identical is that the same primers canbe used to amplify the sequence.

The term “isolated antibody” refers to an antibody that is substantiallyfree of other antibodies having different antigenic specificities (e.g.,an isolated antibody that specifically binds β-klotho is substantiallyfree of antibodies that specifically bind antigens other than 0-klotho).An isolated antibody that specifically binds β-klotho may, however, havecross-reactivity to other antigens. Moreover, an isolated antibody maybe substantially free of other cellular material and/or chemicals.

The term “isotype” refers to the antibody class (e.g., IgM, IgE, IgGsuch as IgG1 or IgG4) that is provided by the heavy chain constantregion genes. Isotype also includes modified versions of one of theseclasses, where modifications have been made to alter the Fc function,for example, to enhance or reduce effector functions or binding to Fcreceptors.

The term “k_(assoc)” or “k_(a)”, as used herein, is intended to refer tothe association rate of a particular antibody-antigen interaction,whereas the term “k_(dis)” or “k_(d),” as used herein, is intended torefer to the dissociation rate of a particular antibody-antigeninteraction. The term “K_(D)”, as used herein, is intended to refer tothe dissociation constant, which is obtained from the ratio of k_(d) tok_(a) (i.e. k_(d)/k_(a)) and is expressed as a molar concentration (M).K_(D) values for antibodies can be determined using methods wellestablished in the art. Methods for determining the K_(D) of an antibodyinclude measuring surface plasmon resonance using a biosensor systemsuch as a Biacore® system, or measuring affinity in solution by solutionequilibrium titration (SET).

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.

The term “nucleic acid” is used herein interchangeably with the term“polynucleotide” and refers to deoxyribonucleotides or ribonucleotidesand polymers thereof in either single- or double-stranded form. The termencompasses nucleic acids containing known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as the reference nucleic acid, and which are metabolized in amanner similar to the reference nucleotides. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides,peptide-nucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Specifically, as detailed below,degenerate codon substitutions may be achieved by generating sequencesin which the third position of one or more selected (or all) codons issubstituted with mixed-base and/or deoxyinosine residues (Batzer et al.,Nucleic Acid Res. 19:5081, 1991; Ohtsuka et al., J. Biol. Chem.260:2605-2608, 1985; and Rossolini et al., Mol. Cell. Probes 8:91-98,1994).

The term “operably linked” refers to a functional relationship betweentwo or more polynucleotide (e.g., DNA) segments. Typically, the termrefers to the functional relationship of a transcriptional regulatorysequence to a transcribed sequence. For example, a promoter or enhancersequence is operably linked to a coding sequence if it stimulates ormodulates the transcription of the coding sequence in an appropriatehost cell or other expression system. Generally, promotertranscriptional regulatory sequences that are operably linked to atranscribed sequence are physically contiguous to the transcribedsequence, i.e., they are cis-acting. However, some transcriptionalregulatory sequences, such as enhancers, need not be physicallycontiguous or located in close proximity to the coding sequences whosetranscription they enhance.

As used herein, the term, “optimized” means that a nucleotide sequencehas been altered to encode an amino acid sequence using codons that arepreferred in the production cell or organism, generally a eukaryoticcell, for example, a cell of Pichia, a Chinese Hamster Ovary cell (CHO)or a human cell. The optimized nucleotide sequence is engineered toretain completely or as much as possible the amino acid sequenceoriginally encoded by the starting nucleotide sequence, which is alsoknown as the “parental” sequence. The optimized sequences herein havebeen engineered to have codons that are preferred in mammalian cells.However, optimized expression of these sequences in other eukaryoticcells or prokaryotic cells is also envisioned herein. The amino acidsequences encoded by optimized nucleotide sequences are also referred toas optimized.

The terms “polypeptide” and “protein” are used interchangeably herein torefer to a polymer of amino acid residues. The terms apply to amino acidpolymers in which one or more amino acid residue is an artificialchemical mimetic of a corresponding naturally occurring amino acid, aswell as to naturally occurring amino acid polymers and non-naturallyoccurring amino acid polymer. Unless otherwise indicated, a particularpolypeptide sequence also implicitly encompasses conservatively modifiedvariants thereof.

The term “recombinant human antibody”, as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as antibodies isolated from an animal (e.g., amouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom, antibodies isolated from a hostcell transformed to express the human antibody, e.g., from atransfectoma, antibodies isolated from a recombinant, combinatorialhuman antibody library, and antibodies prepared, expressed, created orisolated by any other means that involve splicing of all or a portion ofa human immunoglobulin gene, sequences to other DNA sequences. Suchrecombinant human antibodies have variable regions in which theframework and CDR regions are derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies can be subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the VH and VL regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline VH and VL sequences, may not naturally existwithin the human antibody germline repertoire in vivo.

The term “recombinant host cell” (or simply “host cell”) refers to acell into which a recombinant expression vector has been introduced. Itshould be understood that such terms are intended to refer not only tothe particular subject cell but to the progeny of such a cell. Becausecertain modifications may occur in succeeding generations due to eithermutation or environmental influences, such progeny may not, in fact, beidentical to the parent cell, but are still included within the scope ofthe term “host cell” as used herein.

The term “subject” includes human and non-human animals. Non-humananimals include all vertebrates (e.g.: mammals and non-mammals) such as,non-human primates (e.g.: cynomolgus monkey), sheep, dog, cow, chickens,amphibians, and reptiles. Except when noted, the terms “patient” or“subject” are used herein interchangeably. As used herein, the terms“cyno” or “cynomolgus” refer to the cynomolgus monkey (Macacafascicularis).

As used herein, the term “treating” or “treatment” of any disease ordisorder (e.g., FGF21 associated disorder) refers in one embodiment, toameliorating the disease or disorder (i.e., slowing or arresting orreducing the development of the disease or at least one of the clinicalsymptoms thereof). In another embodiment “treating” or “treatment”refers to alleviating or ameliorating at least one physical parameterincluding those which may not be discernible by the patient. In yetanother embodiment, “treating” or “treatment” refers to modulating thedisease or disorder, either physically, (e.g., stabilization of adiscernible symptom), physiologically, (e.g., stabilization of aphysical parameter), or both. In yet another embodiment, “treating” or“treatment” refers to preventing or delaying the onset or development orprogression of the disease or disorder.

“Prevention” as it relates to indications described herein, including,e.g., FGF21 associated disorder, means any action that prevents or slowsa worsening in e.g., FGF21 associated disease parameters, as describedbelow, in a patient at risk for said worsening.

The term “vector” is intended to refer to a polynucleotide moleculecapable of transporting another polynucleotide to which it has beenlinked. One type of vector is a “plasmid”, which refers to a circulardouble stranded DNA loop into which additional DNA segments may beligated. Another type of vector is a viral vector, such as anadeno-associated viral vector (AAV, or AAV2), wherein additional DNAsegments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”).

In general, expression vectors of utility in recombinant DNA techniquesare often in the form of plasmids. In the present specification,“plasmid” and “vector” may be used interchangeably as the plasmid is themost commonly used form of vector. However, the invention is intended toinclude such other forms of expression vectors, such as viral vectors(e.g., replication defective retroviruses, adenoviruses andadeno-associated viruses), which serve equivalent functions.

“Modulation of FGF21 activity,” as used herein, refers to an increase ordecrease in FGF21 activity that can be a result of, for example,interaction of an agent with an FGF21 polynucleotide or polypeptide,activation of the FGF21 signaling pathway and/or activation ofFGF21-mediated signaling (e.g., FGF21-receptor-dependent signaling), andthe like. For example, modulation of a biological activity refers to anincrease or a decrease in a biological activity. FGF21 activity can beassessed by means including, without limitation, assaying blood glucose,insulin, triglyceride, or cholesterol levels in a subject; by assessingpolypeptide levels of beta-klotho and/or FGF receptors (e.g., FGFR-1c);or by assessing activation of FGF21-mediated signaling (e.g., ofFGF21-receptor-dependent signaling).

Comparisons of FGF21 activity can also be accomplished by, e.g.,measuring levels of an FGF21 downstream biomarker, and measuringincreases in FGF21 signaling. Activity can also be assessed bymeasuring: cell signaling; kinase activity; glucose uptake intoadipocytes; blood insulin, triglyceride, or cholesterol levelfluctuations; liver lipid or liver triglyceride level changes;interactions between FGF21 and/or beta-klotho and an FGF21 receptor; orphosphorylation of an FGF21 receptor. In some embodimentsphosphorylation of an FGF21 receptor can be tyrosine phosphorylation. Insome embodiments modulation of FGF21 activity can cause modulation of anFGF21-related phenotype.

An “FGF21 downstream biomarker,” as used herein, is a gene or geneproduct, or measurable indicia of a gene or gene product. In someembodiments, a gene or activity that is a downstream marker of FGF21exhibits an altered level of expression, or in a vascular tissue. Insome embodiments, an activity of the downstream marker is altered in thepresence of an FGF21 modulator. In some embodiments, the downstreammarkers exhibit altered levels of expression when FGF21 is perturbedwith an FGF21 modulator of the present invention. FGF21 downstreammarkers include, without limitation, glucose or 2-deoxy-glucose uptake,pERK and other phosphorylated or acetylated proteins or NAD levels.

As used herein, the term “up-regulates” refers to an increase,activation or stimulation of an activity or quantity. For example, inthe context of the present invention, FGF21 modulators may increase theactivity of beta-klotho and/or an FGF21 receptor. In one embodiment,FGFR-1c may be upregulated in response to an FGF21 modulator.Upregulation can also refer to an FGF21-related activity, such as e.g.,the ability to lower blood glucose, insulin, triglyceride, orcholesterol levels; to reduce liver lipid or triglyceride levels; toreduce body weight; to improve glucose tolerance, energy expenditure, orinsulin sensitivity; or to cause phosphorylation of an FGF21 receptor;or to increase an FGF21 downstream marker. The FGF21 receptor can beβ-klotho and FGFR-1c. Up-regulation may be at least 25%, at least 50%,at least 75%, at least 100%, at least 150%, at least 200%, at least250%, at least 400%, or at least 500% as compared to a control.

As used herein, the term “modulator” refers to a composition thatmodulates one or more physiological or biochemical events associatedwith an FGF21-associated disorder, such as type 1 or type 2 diabetesmellitus or a metabolic condition like obesity. Said events include butare not limited to the ability to lower blood glucose, insulin,triglyceride, or cholesterol levels; to reduce liver lipid or livertriglyceride levels; to reduce body weight; and to improve glucosetolerance, energy expenditure, or insulin sensitivity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-FIG. 1C are graphs showing a solution equilibrium titrationbinding assay of FGF21 mimetic antibodies NOV001, NOV002, and NOV004 tohuman β-klotho.

FIG. 2A and FIG. 2B are graphs showing pERK activation of A) human andB) cynomolgus monkey FGFR1c_β-klotho_HEK293 cells by NOV002 and NOV004.

FIG. 3A-FIG. 3C are graphs showing profiling of NOV002 and NOV004 forpERK activation of human A) FGFR2c_β-klotho, B) FGFR3c_β-klotho, and C)FGFR4_β-klotho HEK293 cells. FGF21 used as a positive control foractivation of FGFR2c_β-klotho or FGFR3c_β-klotho. FGF19 used as positivecontrol for activation of FGFR4_β-klotho.

FIG. 4 is a graph showing profiling of NOV002 and NOV004 for FGF23activity using HEK293 cells transfected with α-klotho, Egr1-luciferaseand Renilla luciferase. FGF23 was used as positive control.

FIG. 5 is a graph showing profiling of NOV002 and NOV004 for mousecross-reactivity using 2-DOG uptake by 3T3-L1 adipocytes. FGF21 was usedas positive control.

FIG. 6 is a graph showing NOV002 and NOV004 concentration-time profilesfollowing IV injection in rats.

DETAILED DESCRIPTION

The present invention is based, in part, on the discovery of antibodymolecules that specifically bind to β-klotho and lead to activation ofFGF receptors, e.g., FGFR1c, and the activation of FGF21-mediatedsignaling (e.g., FGF21-receptor-dependent signaling). The inventionrelates to both full IgG format antibodies as well as antigen-bindingfragments thereof, such as Fab fragments (e.g., antibodies NOV001,NOV002, NOV003, and NOV004).

Accordingly, the present invention provides antibodies that specificallybind to β-klotho (e.g., human and cynomolgus monkey β-klotho),pharmaceutical compositions, production methods, and methods of use ofsuch antibodies and compositions.

FGF21 Proteins

The present disclosure provides FGF21 mimetic mAbs (i.e., monoclonalantibodies that bind to beta-klotho (β-klotho)) that can induceFGF21-mediated signaling (e.g., FGF21-receptor-mediated signaling), asdefined herein. In vivo, the mature form of FGF21 is the active form ofthe molecule. The FGF21 wild-type sequence has NCBI reference sequencenumber NP_061986.1, and can be found in such issued patents as, e.g.,U.S. Pat. No. 6,716,626 B1, assigned to Chiron Corporation (SEQ IDNO:1).

Met Asp Ser Asp Glu Thr Gly Phe Glu His Ser Gly Leu Trp Val Ser 1               5                  10                  15Val Leu Ala Gly Leu Leu Leu Gly Ala Cys Gln Ala His Pro Ile Pro            20                  25                  30Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr        35                  40                  45Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg    50                  55                  60Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu65                  70                  75                  80Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu Gly Val                85                  90                  95Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly            100                 105                 110Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu        115                 120                 125Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu    130                 135                 140His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly145                 150                 155                 160Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Leu Pro Glu                165                 170                 175Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp            180                 185                 190Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser Tyr Ala        195                 200                 205 Ser 209

The corresponding mRNA sequence coding for the full-length FGF21polypeptide (NCBI reference sequence number NM_019113.2) is shown below(SEQ ID NO:2)

1 ctgtcagctg aggatccagc cgaaagagga gccaggcact caggccacct gagtctactc 61acctggacaa ctggaatctg gcaccaattc taaaccactc agcttctccg agctcacacc 121ccggagatca cctgaggacc cgagccattg atggactcgg acgagaccgg gttcgagcac 181tcaggactgt gggtttctgt gctggctggt cttctgctgg gagcctgcca ggcacacccc 241atccctgact ccagtcctct cctgcaattc gggggccaag tccggcagcg gtacctctac 301acagatgatg cccagcagac agaagcccac ctggagatca gggaggatgg gacggtgggg 361ggcgctgctg accagagccc cgaaagtctc ctgcagctga aagccttgaa gccgggagtt 421attcaaatct tgggagtcaa gacatccagg ttcctgtgcc agcggccaga tggggccctg 481tatggatcgc tccactttga ccctgaggcc tgcagcttcc gggagctgct tcttgaggac 541ggatacaatg tttaccagtc cgaagcccac ggcctcccgc tgcacctgcc agggaacaag 601tccccacacc gggaccctgc accccgagga ccagctcgct tcctgccact accaggcctg 661ccccccgcac tcccggagcc acccggaatc ctggcccccc agccccccga tgtgggctcc 721tcggaccctc tgagcatggt gggaccttcc cagggccgaa gccccagcta cgcttcctga 781agccagaggc tgtttactat gacatctcct ctttatttat taggttattt atcttattta 841tttttttatt tttcttactt gagataataa agagttccag aggagaaaaa aaaaaaaaaa 901aaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

The mature FGF21 sequence lacks a leader sequence and may also includeother modifications of a polypeptide such as proteolytic processing ofthe amino terminus (with or without a leader sequence) and/or thecarboxyl terminus, cleavage of a smaller polypeptide from a largerprecursor, N-linked and/or O-linked glycosylation, and otherpost-translational modifications understood by those with skill in theart. A representative example of a mature FGF21 sequence has thefollowing sequence (SEQ ID NO:83, which represents amino acid positions29-209 of full length FGF21 protein sequence (NCBI reference sequencenumber NP_061986.1)):

His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val                 5              10                      15Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His            20               25                      30Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser         35                  40                  45Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln     50                  55                  60Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly 65                  70                  75                  80Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg                 85                  90                  95Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His            100                 105                 110Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro        115                 120                 125Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro    130                 135                 140Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val145                 150                 155                 160Gly Ser Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser                165                 170                 175Pro Ser Tyr Ala Ser             180

The corresponding cDNA sequence coding for the mature FGF21 polypeptide(SEQ ID NO:83) is shown below (SEQ ID NO:84):

1 caccccatcc ctgactccag tcctctcctg caattcgggg gccaagtccg gcagcggtac 61ctctacacag atgatgccca gcagacagaa gcccacctgg agatcaggga ggatgggacg 121gtggggggcg ctgctgacca gagccccgaa agtctcctgc agctgaaagc cttgaagccg 181ggagttattc aaatcttggg agtcaagaca tccaggttcc tgtgccagcg gccagatggg 240gccctgtatg gatcgctcca ctttgaccct gaggcctgca gcttccggga gctgcttctt 301gaggacggat acaatgttta ccagtccgaa gcccacggcc tcccgctgca cctgccaggg 360aacaagtccc cacaccggga ccctgcaccc cgaggaccag ctcgcttcct gccactacca 421ggcctgcccc ccgcactccc ggagccaccc ggaatcctgg ccccccagcc ccccgatgtg 481ggctcctcgg accctctgag catggtggga ccttcccagg gccgaagccc cagctacgct 541tcctgaFGF21 Mimetic Antibodies & Antigen-Binding Fragments

The present invention provides antibodies that specifically bind toβ-klotho. In some embodiments, the present invention provides antibodiesthat specifically bind to human and cynomolgus monkey β-klotho.Antibodies of the invention include, but are not limited to, the humanmonoclonal antibodies and Fabs, isolated as described in the Examples.

The β-klotho wild-type sequence has NCBI reference sequence numberNP_783864.1, and can be found in such literature as Xu, et al. (2007) JBiol Chem. 282(40):29069-72 and Lin, et al. (2007) J Biol Chem.282(37):27277-84. The full-length cDNA encoding human β-klotho hasGenBank Accession number NM_175737). The protein sequence is as follows(SEQ ID NO:262).

1 mkpgcaagsp gnewiffstd eittryrntm sngglqrsvi lsalillrav tgfsgdgrai 61wsknpnftpv nesqlflydt fpknffwgig tgalqvegsw kkdgkgpsiw dhfihthlkn 121vsstngssds yiflekdlsa ldfigvsfyq fsiswprlfp dgivtvanak glqyystlld 181alvlrniepi vtlyhwdlpl alqekyggwk ndtiidifnd yatycfqmfg drvkywitih 241npylvawhgy gtgmhapgek gnlaavytvg hnlikahskv whnynthfrp hqkgwlsitl 301gshwiepnrs entmdifkcq qsmvsvlgwf anpihgdgdy pegmrkklfs vlpifseaek 361hemrgtadff afsfgpnnfk pintmakmgq nvslnlreal nwikleynnp riliaengwf 421tdsrvktedt taiymmknfl sqvlqairld eirvfgytaw slldgfewqd aytirrglfy 481vdfnskqker kpkssahyyk qiirengfsl kestpdvqgq fpcdfswgvt esvlkpesva 541sspqfsdphl yvwnatgnrl lhrvegvrlk trpaqctdfv nikkqlemla rmkvthyrfa 601ldwasvlptg nlsavnrgal ryyrcvvseg lklgisamvt lyypthahlg lpepllhadg 661wlnpstaeaf qayaglcfge lgdlvklwit inepnrlsdi ynrsgndtyg aahnllvaha 721lawrlydrqf rpsqrgaysl slhadwaepa npyadshwra aerflqfeia wfaeplfktg 781dypaamreyi askhrrglss salprlteae rrllkgtvdf calnhfttrf vmheqlagsr 841ydsdrdiqfl qditrlsspt rlavipwgvr kllrwvrrny gdmdiyitas giddqaledd 901rlrkyylgky lqevlkayli dkvrikgyya fklaeekskp rfgfftsdfk akssiqfynk 961vissrgfpfe nsssrcsqtq entectvclf lvqkkplifl gccffstivl llsiaifqrq 1021krrkfwkakn lqhiplkkgk rvvs

The present invention provides antibodies that specifically bind aβ-klotho protein (e.g., human and cynomolgus monkey β-klotho), whereinthe antibodies comprise a VH domain having an amino acid sequence of SEQID NOs: 9, 29, 49, or 69. The present invention also provides antibodiesthat specifically bind to a β-klotho protein, wherein the antibodiescomprise a VH CDR having an amino acid sequence of any one of the VHCDRs listed in Table 1, infra. In particular, the invention providesantibodies that specifically bind to a β-klotho protein (e.g., human andcynomolgus monkey β-klotho), wherein the antibodies comprise (oralternatively, consist of) one, two, three, or more VH CDRs having anamino acid sequence of any of the VH CDRs listed in Table 1, infra.

The present invention provides antibodies that specifically bind to aβ-klotho protein, said antibodies comprising a VL domain having an aminoacid sequence of SEQ ID NOs: 19, 39, 59, or 79. The present inventionalso provides antibodies that specifically bind to a β-klotho protein(e.g., human and cynomolgus monkey β-klotho), said antibodies comprisinga VL CDR having an amino acid sequence of any one of the VL CDRs listedin Table 1, infra. In particular, the invention provides antibodies thatspecifically bind to a β-klotho protein (e.g., human and cynomolgusmonkey β-klotho), said antibodies comprising (or alternatively,consisting of) one, two, three or more VL CDRs having an amino acidsequence of any of the VL CDRs listed in Table 1, infra.

Other antibodies of the invention include amino acids that have beenmutated, yet have at least 60, 70, 80, 85, 90 or 95 percent identity inthe CDR regions with the CDR regions depicted in the sequences describedin Table 1. In some embodiments, it includes mutant amino acid sequenceswherein no more than 1, 2, 3, 4 or 5 amino acids have been mutated inthe CDR regions when compared with the CDR regions depicted in thesequence described in Table 1.

The present invention also provides nucleic acid sequences that encodeVH, VL, the full length heavy chain, and the full length light chain ofthe antibodies that specifically bind to a β-klotho protein (e.g., humanand cynomolgus monkey β-klotho). Such nucleic acid sequences can beoptimized for expression in mammalian cells (for example, Table 1 showsthe optimized nucleic acid sequences for the heavy chain and light chainof antibodies of the invention).

TABLE 1 Examples of FGF21 Mimetic Antibodies and Fabs Sequence SequenceIdentifier Description (SEQ ID NO:)Amino acid or polynucleotide sequence NOV001 (IgG1 LALA version ofNOV003) HCDR1 (Kabat) 3 DYYIN HCDR2 (Kabat) 4 RIHPGSGNTYYNEKFQGHCDR3 (Kabat) 5 LLLRSYGMDD HCDR1 (Chothia) 6 GYTFTDY HCDR2 (Chothia) 7HPGSGN HCDR3 (Chothia) 8 LLLRSYGMDD HCDR1 (Combined) 263 GYTFTDYYINHCDR2 (Combined) 4 RIHPGSGNTYYNEKFQG HCDR3 (Combined) 5 LLLRSYGMDDHCDR1 (IMGT) 264 GYTFTDYY HCDR2 (IMGT) 265 IHPGSGNT HCDR3 (IMGT) 266AILLLRSYGMDD VH 9 QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYYINWVRQAPGQGLEWMGRIHPGSGNTYYNEKFQGRVTLTADKSTSTAYMELSSLRSEDTAVYYCAILLLRSYGMDDWGQGTTVTVSS DNA VH 10CAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCCGGCAGCAGCGTGAAGGTGTCCTGCAAGGCCAGCGGCTACACCTTTACCGACTACTACATCAACTGGGTGCGCCAGGCCCCTGGACAGGGCCTGGAATGGATGGGCAGAATCCACCCCGGCTCCGGCAACACCTACTACAACGAGAAGTTCCAGGGCAGAGTGACCCTGACCGCCGACAAGAGCACCAGCACCGCCTACATGGAACTGAGCAGCCTGCGGAGCGAGGACACCGCCGTGTACTACTGTGCCATCCTGCTGCTGCGGAGCTACGGCATGGATGATTGGGGCCAGGGCACCACCGTGACCGTCAGCTCA Heavy Chain 11QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYYINWVRQAPGQGLEWMGRIHPGSGNTYYNEKFQGRVTLTADKSTSTAYMELSSLRSEDTAVYYCAILLLRSYGMDDWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQ KSLSLSPGK DNA Heavy Chain 12CAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCCGGCAGCAGCGTGAAGGTGTCCTGCAAGGCCAGCGGCTACACCTTTACCGACTACTACATCAACTGGGTGCGCCAGGCCCCTGGACAGGGCCTGGAATGGATGGGCAGAATCCACCCCGGCTCCGGCAACACCTACTACAACGAGAAGTTCCAGGGCAGAGTGACCCTGACCGCCGACAAGAGCACCAGCACCGCCTACATGGAACTGAGCAGCCTGCGGAGCGAGGACACCGCCGTGTACTACTGTGCCATCCTGCTGCTGCGGAGCTACGGCATGGATGATTGGGGCCAGGGCACCACCGTGACCGTCAGCTCAGCTAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGTCCAGCGTGGTGACAGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCAGCCCCAGAGGCAGCGGGCGGACCCTCCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCAGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGCAAGGTCTCCAACAAGGCCCTGCCAGCCCCCATCGAAAAGACCATCAGCAAGGCCAAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGCCCCCCTCCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAG AAGAGCCTGAGCCTGTCCCCCGGCAAGLCDR1 (Kabat) 13 KSSQSIVHSSGNTYLE LCDR2 (Kabat) 14 KVSNRFS LCDR3 (Kabat)15 FQGSHIPYT LCDR1 (Chothia) 16 SQSIVHSSGNTY LCDR2 (Chothia) 17 KVSLCDR3 (Chothia) 18 GSHIPY LCDR1 (Combined) 13 KSSQSIVHSSGNTYLELCDR2 (Combined) 14 KVSNRFS LCDR3 (Combined) 15 FQGSHIPYT LCDR1 (IMGT)267 QSIVHSSGNTY LCDR2 (IMGT) 17 KVS LCDR3 (IMGT) 15 FQGSHIPYT VL 19DVVMTQTPLSLSVTPGQPASISCKSSQSIVHSSGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIK DNA VL 20GACGTGGTGATGACCCAGACCCCCCTGAGCCTGAGCGTGACACCTGGACAGCCTGCCAGCATCTCCTGCAAGAGCAGCCAGAGCATCGTGCACAGCAGCGGCAACACCTACCTGGAATGGTATCTGCAGAAGCCCGGCCAGAGCCCCCAGCTGCTGATCTACAAGGTGTCCAACCGGTTCAGCGGCGTGCCCGACAGATTTTCTGGCAGCGGCTCCGGCACCGACTTCACCCTGAAGATCTCCCGGGTGGAAGCCGAGGACGTGGGCGTGTACTACTGTTTTCAAGGCTCCCACATCCCCTACACCTTCGGCCAGGGCAC CAAGCTGGAAATCAAG Light Chain 21DVVMTQTPLSLSVTPGQPASISCKSSQSIVHSSGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGECDNA Light Chain 22 GACGTGGTGATGACCCAGACCCCCCTGAGCCTGAGCGTGACACCTGGACAGCCTGCCAGCATCTCCTGCAAGAGCAGCCAGAGCATCGTGCACAGCAGCGGCAACACCTACCTGGAATGGTATCTGCAGAAGCCCGGCCAGAGCCCCCAGCTGCTGATCTACAAGGTGTCCAACCGGTTCAGCGGCGTGCCCGACAGATTTTCTGGCAGCGGCTCCGGCACCGACTTCACCCTGAAGATCTCCCGGGTGGAAGCCGAGGACGTGGGCGTGTACTACTGTTTTCAAGGCTCCCACATCCCCTACACCTTCGGCCAGGGCACCAAGCTGGAAATCAAGCGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCT TCAACAGGGGCGAGTGC NOV002(IgG1 LALA version of NOV004) HCDR1 (Kabat) 23 SGYTWH HCDR2 (Kabat) 24YIHYSVYTNYNPSVKG HCDR3 (Kabat) 25 RTTSLERYFDV HCDR1 (Chothia) 26GYSITSGY HCDR2 (Chothia) 27 HYSVY HCDR3 (Chothia) 28 RTTSLERYFDVHCDR1 (Combined) 268 GYSITSGYTWH HCDR2 (Combined) 24 YIHYSVYTNYNPSVKGHCDR3 (Combined) 25 RTTSLERYFDV HCDR1 (IMGT) 269 GYSITSGYT HCDR2 (IMGT)270 IHYSVYT HCDR3 (IMGT) 271 ARRTTSLERYFDV VH 29EVQLVESGGGLVKPGGSLRLSCAVSGYSITSGYTWHWVRQAPGKGLEWLSYIHYSVYTNYNPSVKGRFTISRDTAKNSFYLQMNSLRAEDTAVYYCARRTTSLERYFDVWGQGTLVTVSS DNA VH 30GAGGTGCAGCTGGTGGAATCTGGCGGCGGACTCGTGAAGCCTGGCGGCTCTCTGAGACTGAGCTGTGCCGTGTCCGGCTACAGCATCACCAGCGGCTACACCTGGCATTGGGTGCGCCAGGCCCCTGGCAAAGGACTGGAATGGCTGTCCTACATCCACTACAGCGTGTACACCAACTACAACCCCAGCGTGAAGGGCCGGTTCACCATCAGCAGAGACACCGCCAAGAACAGCTTCTACCTGCAAATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTGTGCCAGACGGACCACCAGCCTGGAACGGTACTTCGACGTGTGGGGCCAGGGCACACTCGTGACCGTCAGCTCA Heavy Chain 31EVQLVESGGGLVKPGGSLRLSCAVSGYSITSGYTWHWVRQAPGKGLEWLSYIHYSVYTNYNPSVKGRFTISRDTAKNSFYLQMNSLRAEDTAVYYCARRTTSLERYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEALHNHYT QKSLSLSPGK DNA Heavy Chain 32GAGGTGCAGCTGGTGGAATCTGGCGGCGGACTCGTGAAGCCTGGCGGCTCTCTGAGACTGAGCTGTGCCGTGTCCGGCTACAGCATCACCAGCGGCTACACCTGGCATTGGGTGCGCCAGGCCCCTGGCAAAGGACTGGAATGGCTGTCCTACATCCACTACAGCGTGTACACCAACTACAACCCCAGCGTGAAGGGCCGGTTCACCATCAGCAGAGACACCGCCAAGAACAGCTTCTACCTGCAAATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTGTGCCAGACGGACCACCAGCCTGGAACGGTACTTCGACGTGTGGGGCCAGGGCACACTCGTGACCGTCAGCTCAGCTAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGTCCAGCGTGGTGACAGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCAGCCCCAGAGGCAGCGGGCGGACCCTCCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCAGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGCAAGGTCTCCAACAAGGCCCTGCCAGCCCCCATCGAAAAGACCATCAGCAAGGCCAAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGCCCCCCTCCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACC CAGAAGAGCCTGAGCCTGTCCCCCGGCAAGLCDR1 (Kabat) 33 QASQDISNYLN LCDR2 (Kabat) 34 YTSRLQS LCDR3 (Kabat) 35QQGNTLPYT LCDR1 (Chothia) 36 SQDISNY LCDR2 (Chothia) 37 YTSLCDR3 (Chothia) 38 GNTLPY LCDR1 (Combined) 33 QASQDISNYLNLCDR2 (Combined) 34 YTSRLQS LCDR3 (Combined) 35 QQGNTLPYT LCDR1 (IMGT)272 QDISNY LCDR2 (IMGT) 273 YTS LCDR3 (IMGT) 35 QQGNTLPYT VL 39DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYYTSRLQSGVPSRFTGSGSGADYTFTISSLQP EDIATYFCQQGNTLPYTFGQGTKLEIKDNA VL 40 GACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGTGACCATCACCTGTCAGGCCAGCCAGGACATCAGCAACTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACTACACCAGCAGACTGCAGAGCGGCGTGCCCAGCAGATTTACCGGCTCTGGAAGCGGAGCCGACTACACCTTCACCATCAGCTCCCTGCAGCCCGAGGATATCGCTACCTACTTCTGTCAGCAAGGCAACACCCTGCCTTACACCTTCGGCCAGGGCACCAAGCTGGAAATCAA G Light Chain 41DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYYTSRLQSGVPSRFTGSGSGADYTFTISSLQPEDIATYFCQQGNTLPYTEGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC DNA Light Chain42 GACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGTGACCATCACCTGTCAGGCCAGCCAGGACATCAGCAACTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACTACACCAGCAGACTGCAGAGCGGCGTGCCCAGCAGATTTACCGGCTCTGGAAGCGGAGCCGACTACACCTTCACCATCAGCTCCCTGCAGCCCGAGGATATCGCTACCTACTTCTGTCAGCAAGGCAACACCCTGCCTTACACCTTCGGCCAGGGCACCAAGCTGGAAATCAAGCGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACAGGGGCGAGT GC NOV003 (IgG1 DAPA version ofNOV001) HCDR1 (Kabat) 43 DYYIN HCDR2 (Kabat) 44 RIHPGSGNTYYNEKFQGHCDR3 (Kabat) 45 LLLRSYGMDD HCDR1 (Chothia) 46 GYTFTDY HCDR2 (Chothia)47 HPGSGN HCDR3 (Chothia) 48 LLLRSYGMDD HCDR1 (Combined) 263 GYTFTDYYINHCDR2 (Combined) 44 RIHPGSGNTYYNEKFQG HCDR3 (Combined) 45 LLLRSYGMDDHCDR1 (IMGT) 264 GYTFTDYY HCDR2 (IMGT) 265 IHPGSGNT HCDR3 (IMGT) 266AILLLRSYGMDD VH 49 QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYYINWVRQAPGQGLEWMGRIHPGSGNTYYNEKFQGRVTLTADKSTSTAYMELSSLRSEDTAVYYCAILLLRSYGMDDWGQGTTVTVSS DNA VH 50CAAGTCCAACTCGTCCAGTCCGGAGCCGAAGTGAAAAAGCCGGGCTCATCAGTGAAGGTGTCCTGCAAGGCGTCGGGCTACACCTTCACCGACTACTACATCAACTGGGTGCGCCAGGCCCCGGGACAGGGTCTGGAATGGATGGGGAGGATTCACCCCGGATCGGGAAACACCTACTACAACGAGAAGTTCCAGGGCAGAGTGACCCTGACTGCCGACAAGTCCACGTCCACTGCCTACATGGAACTGTCGTCCCTGCGGTCCGAGGATACCGCCGTGTACTATTGTGCGATCCTGCTGTTGCGGAGCTACGGGATGGATGACTGGGGACAGGGTACCACTGTGACTGTGTCCAGC Heavy Chain 51QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYYINWVRQAPGQGLEWMGRIHPGSGNTYYNEKFQGRVTLTADKSTSTAYMELSSLRSEDTAVYYCAILLLRSYGMDDWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQ KSLSLSPGK DNA Heavy Chain 52CAAGTCCAACTCGTCCAGTCCGGAGCCGAAGTGAAAAAGCCGGGCTCATCAGTGAAGGTGTCCTGCAAGGCGTCGGGCTACACCTTCACCGACTACTACATCAACTGGGTGCGCCAGGCCCCGGGACAGGGTCTGGAATGGATGGGGAGGATTCACCCCGGATCGGGAAACACCTACTACAACGAGAAGTTCCAGGGCAGAGTGACCCTGACTGCCGACAAGTCCACGTCCACTGCCTACATGGAACTGTCGTCCCTGCGGTCCGAGGATACCGCCGTGTACTATTGTGCGATCCTGCTGTTGCGGAGCTACGGGATGGATGACTGGGGACAGGGTACCACTGTGACTGTGTCCAGCGCTAGCACCAAGGGCCCCTCCGTGTTCCCTCTGGCCCCTTCCAGCAAGTCTACCTCCGGCGGCACAGCTGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCTGAGCCTGTGACAGTGTCCTGGAACTCTGGCGCCCTGACCTCTGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTGGTCACAGTGCCTTCAAGCAGCCTGGGCACCCAGACCTATATCTGCAACGTGAACCACAAGCCTTCCAACACCAAGGTGGACAAGCGGGTGGAGCCTAAGTCCTGCGACAAGACCCACACCTGTCCTCCCTGCCCTGCTCCTGAACTGCTGGGCGGCCCTTCTGTGTTCCTGTTCCCTCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCTGAAGTGACCTGCGTGGTGGTGGCCGTGTCCCACGAGGATCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAAGTCTCCAACAAGGCCCTGGCCGCCCCTATCGAAAAGACAATCTCCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTGTACACCCTGCCACCCAGCCGGGAGGAAATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGTGGAGTGGGAGTCTAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCTGTGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCAAACTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAG AAGTCCCTGTCCCTGTCTCCCGGCAAGLCDR1 (Kabat) 53 KSSQSIVHSSGNTYLE LCDR2 (Kabat) 54 KVSNRFS LCDR3 (Kabat)55 FQGSHIPYT LCDR1 (Chothia) 56 SQSIVHSSGNTY LCDR2 (Chothia) 57 KVSLCDR3 (Chothia) 58 GSHIPY LCDR1 (Combined) 53 KSSQSIVHSSGNTYLELCDR2 (Combined) 54 KVSNRFS LCDR3 (Combined) 55 FQGSHIPYT LCDR1 (IMGT)267 QSIVHSSGNTY LCDR2 (IMGT) 57 KVS LCDR3 (IMGT) 55 FQGSHIPYT VL 59DVVMTQTPLSLSVTPGQPASISCKSSQSIVHSSGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIK DNA VL 60GATGTCGTGATGACCCAGACTCCGCTGTCCCTGTCCGTGACCCCTGGACAGCCCGCGTCTATCTCGTGCAAGAGCTCCCAGTCCATTGTGCATTCAAGCGGGAACACCTATCTGGAGTGGTACCTCCAGAAGCCTGGCCAGAGCCCACAGCTGCTGATCTACAAAGTGTCGAACAGATTCTCCGGTGTCCCGGACCGGTTCTCCGGCTCGGGAAGCGGCACTGACTTTACACTGAAGATCTCACGGGTGGAAGCCGAGGACGTGGGAGTGTACTACTGTTTCCAAGGGTCCCACATTCCCTACACCTTCGGCCAAGGAAC TAAGCTGGAAATCAAG Light Chain 61DVVMTQTPLSLSVTPGQPASISCKSSQSIVHSSGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGECDNA Light Chain 62 GATGTCGTGATGACCCAGACTCCGCTGTCCCTGTCCGTGACCCCTGGACAGCCCGCGTCTATCTCGTGCAAGAGCTCCCAGTCCATTGTGCATTCAAGCGGGAACACCTATCTGGAGTGGTACCTCCAGAAGCCTGGCCAGAGCCCACAGCTGCTGATCTACAAAGTGTCGAACAGATTCTCCGGTGTCCCGGACCGGTTCTCCGGCTCGGGAAGCGGCACTGACTTTACACTGAAGATCTCACGGGTGGAAGCCGAGGACGTGGGAGTGTACTACTGTTTCCAAGGGTCCCACATTCCCTACACCTTCGGCCAAGGAACTAAGCTGGAAATCAAGCGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCT TCAACAGGGGCGAGTGC NOV004(IgG1 DAPA version of NOV002) HCDR1 (Kabat) 63 SGYTWH HCDR2 (Kabat) 64YIHYSVYTNYNPSVKG HCDR3 (Kabat) 65 RTTSLERYFDV HCDR1 (Chothia) 66GYSITSGY HCDR2 (Chothia) 67 HYSVY HCDR3 (Chothia) 68 RTTSLERYFDVHCDR1 (Combined) 268 GYSITSGYTWH HCDR2 (Combined) 64 YIHYSVYTNYNPSVKGHCDR3 (Combined) 25 RTTSLERYFDV HCDR1 (IMGT) 269 GYSITSGYT HCDR2 (IMGT)270 IHYSVYT HCDR3 (IMGT) 271 ARRTTSLERYFDV VH 69EVQLVESGGGLVKPGGSLRLSCAVSGYSITSGYTWHWVRQAPGKGLEWLSYIHYSVYTNYNPSVKGRFTISRDTAKNSFYLQMNSLRAEDTAVYYCARRTTSLERYFDVWGQGTLVTVSS DNA VH 70GAAGTCCAACTCGTCGAATCCGGCGGCGGACTGGTCAAGCCGGGAGGATCGCTGAGACTGTCGTGCGCAGTGTCAGGGTACAGCATCACCTCCGGTTACACCTGGCACTGGGTCAGACAGGCGCCGGGAAAAGGCCTGGAATGGCTGTCCTACATTCATTACTCCGTGTACACTAACTACAACCCCTCAGTGAAGGGGCGGTTCACCATCTCCCGGGACACTGCCAAGAATAGCTTCTATCTGCAAATGAACTCCCTGCGGGCCGAGGATACCGCCGTGTACTACTGCGCGAGGCGCACCACGTCCCTGGAGCGCTACTTTGACGTGTGGGGCCAGGGTACCCTCGTGACTGTGTCCTCG Heavy Chain 71EVQLVESGGGLVKPGGSLRLSCAVSGYSITSGYTWHWVRQAPGKGLEWLSYIHYSVYTNYNPSVKGRFTISRDTAKNSFYLQMNSLRAEDTAVYYCARRTTSLERYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEALHNHYT QKSLSLSPGK DNA Heavy Chain 72GAAGTCCAACTCGTCGAATCCGGCGGCGGACTGGTCAAGCCGGGAGGATCGCTGAGACTGTCGTGCGCAGTGTCAGGGTACAGCATCACCTCCGGTTACACCTGGCACTGGGTCAGACAGGCGCCGGGAAAAGGCCTGGAATGGCTGTCCTACATTCATTACTCCGTGTACACTAACTACAACCCCTCAGTGAAGGGGCGGTTCACCATCTCCCGGGACACTGCCAAGAATAGCTTCTATCTGCAAATGAACTCCCTGCGGGCCGAGGATACCGCCGTGTACTACTGCGCGAGGCGCACCACGTCCCTGGAGCGCTACTTTGACGTGTGGGGCCAGGGTACCCTCGTGACTGTGTCCTCGGCTAGCACCAAGGGCCCCTCCGTGTTCCCTCTGGCCCCTTCCAGCAAGTCTACCTCCGGCGGCACAGCTGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCTGAGCCTGTGACAGTGTCCTGGAACTCTGGCGCCCTGACCTCTGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTGGTCACAGTGCCTTCAAGCAGCCTGGGCACCCAGACCTATATCTGCAACGTGAACCACAAGCCTTCCAACACCAAGGTGGACAAGCGGGTGGAGCCTAAGTCCTGCGACAAGACCCACACCTGTCCTCCCTGCCCTGCTCCTGAACTGCTGGGCGGCCCTTCTGTGTTCCTGTTCCCTCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCTGAAGTGACCTGCGTGGTGGTGGCCGTGTCCCACGAGGATCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAAGTCTCCAACAAGGCCCTGGCCGCCCCTATCGAAAAGACAATCTCCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTGTACACCCTGCCACCCAGCCGGGAGGAAATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGTGGAGTGGGAGTCTAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCTGTGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCAAACTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACC CAGAAGTCCCTGTCCCTGTCTCCCGGCAAGLCDR1 (Kabat) 73 QASQDISNYLN LCDR2 (Kabat) 74 YTSRLQS LCDR3 (Kabat) 75QQGNTLPYT LCDR1 (Chothia) 76 SQDISNY LCDR2 (Chothia) 77 YTSLCDR3 (Chothia) 78 GNTLPY LCDR1 (Combined) 73 QASQDISNYLNLCDR2 (Combined) 74 YTSRLQS LCDR3 (Combined) 75 QQGNTLPYT LCDR1 (IMGT)272 QDISNY LCDR2 (IMGT) 273 YTS LCDR3 (IMGT) 75 QQGNTLPYT VL 79DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYYTSRLQSGVPSRFTGSGSGADYTFTISSLQP EDIATYFCQQGNTLPYTFGQGTKLEIKDNA VL 80 GATATTCAGATGACTCAGAGCCCCTCCTCGCTCTCCGCCTCCGTGGGGGATCGCGTGACAATCACCTGTCAAGCGTCCCAGGACATCTCAAACTACCTGAACTGGTATCAGCAGAAGCCAGGGAAGGCCCCGAAGCTGCTGATCTACTACACTTCGCGGCTGCAGTCCGGCGTGCCGTCACGGTTCACTGGCTCGGGCTCCGGAGCAGACTACACCTTCACCATTAGCAGCCTGCAGCCCGAGGACATCGCTACCTACTTTTGCCAACAAGGAAACACCCTGCCTTACACCTTCGGACAGGGTACTAAGCTGGAAATCAA A Light Chain 81DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYYTSRLQSGVPSRFTGSGSGADYTFTISSLQPEDIATYFCQQGNTLPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC DNA Light Chain82 GATATTCAGATGACTCAGAGCCCCTCCTCGCTCTCCGCCTCCGTGGGGGATCGCGTGACAATCACCTGTCAAGCGTCCCAGGACATCTCAAACTACCTGAACTGGTATCAGCAGAAGCCAGGGAAGGCCCCGAAGCTGCTGATCTACTACACTTCGCGGCTGCAGTCCGGCGTGCCGTCACGGTTCACTGGCTCGGGCTCCGGAGCAGACTACACCTTCACCATTAGCAGCCTGCAGCCCGAGGACATCGCTACCTACTTTTGCCAACAAGGAAACACCCTGCCTTACACCTTCGGACAGGGTACTAAGCTGGAAATCAAACGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACAGGGGCGAGT GC

Other antibodies of the invention include those where the amino acids ornucleic acids encoding the amino acids have been mutated, yet have atleast 60, 65, 70, 75, 80, 85, 90, or 95 percent identity to thesequences described in Table 1. Some embodiments include mutant aminoacid sequences wherein no more than 1, 2, 3, 4 or 5 amino acids havebeen mutated in the variable regions when compared with the variableregions depicted in the sequence described in Table 1, while retainingsubstantially the same antigen-binding activity.

Other antibodies of the invention include those where the amino acids ornucleic acids encoding the amino acids have been mutated, yet have atleast 60, 65, 70, 75, 80, 85, 90, or 95 percent identity to thesequences described in Table 1. Some embodiments include mutant aminoacid sequences wherein no more than 1, 2, 3, 4 or 5 amino acids havebeen mutated in the variable regions when compared with the variableregions depicted in the sequence described in Table 1, while retainingsubstantially the same antigen-binding activity.

Since each of these antibodies can bind to β-klotho, the VH, VL, fulllength light chain, and full length heavy chain sequences (amino acidsequences and the nucleotide sequences encoding the amino acidsequences) can be “mixed and matched” to create other β-klotho-bindingantibodies of the invention. Such “mixed and matched”—β-klotho bindingantibodies can be tested using the binding assays known in the art(e.g., ELISAs, and other assays described in the Example section). Whenthese chains are mixed and matched, a VH sequence from a particularVH/VL pairing should be replaced with a structurally similar VHsequence. Likewise a full length heavy chain sequence from a particularfull length heavy chain/full length light chain pairing should bereplaced with a structurally similar full length heavy chain sequence.Likewise, a VL sequence from a particular VH/VL pairing should bereplaced with a structurally similar VL sequence. Likewise a full lengthlight chain sequence from a particular full length heavy chain/fulllength light chain pairing should be replaced with a structurallysimilar full length light chain sequence.

Accordingly, in one aspect, the invention provides an isolated antibodyor antigen-binding region thereof having: a heavy chain variable domaincomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 9, 29, 49, and 69, and a light chain variable domaincomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 19, 39, 59, and 79, wherein the antibody specifically bindsto β-klotho (e.g., human and cynomolgus monkey β-klotho).

More specifically, in certain aspects, the invention provides anisolated antibody or antigen-binding region thereof having a heavy chainvariable domain and a light chain variable domain comprising amino acidsequences selected from SEQ ID NOs: 9 and 19; 29 and 39; 49 and 59; or69 and 79, respectively.

In another aspect, the invention provides (i) an isolated antibodyhaving: a full length heavy chain comprising an amino acid sequence thathas been optimized for expression in a mammalian cell selected from thegroup consisting of SEQ ID NOs: 9, 29, 49, or 69, and a full lengthlight chain comprising an amino acid sequence that has been optimizedfor expression in a mammalian cell selected from the group consisting ofSEQ ID NOs: 21, 41, 61, or 81; or (ii) a functional protein comprisingan antigen-binding portion thereof. More specifically, in certainaspects, the invention provides an isolated antibody or antigen-bindingregion thereof having a heavy chain and a light chain comprising aminoacid sequences selected from SEQ ID NOs: 9 and 19; 29 and 39; 49 and 59;or 69 and 79, respectively.

The terms “complementarity determining region,” and “CDR,” as usedherein refer to the sequences of amino acids within antibody variableregions which confer antigen specificity and binding affinity. Ingeneral, there are three CDRs in each heavy chain variable region(HCDR1, HCDR2, HCDR3) and three CDRs in each light chain variable region(LCDR1, LCDR2, LCDR3).

The precise amino acid sequence boundaries of a given CDR can be readilydetermined using any of a number of well-known schemes, including thosedescribed by Kabat et al. (1991), “Sequences of Proteins ofImmunological Interest,” 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (“Kabat” numbering scheme),Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numberingscheme), and ImMunoGenTics (IMGT) numbering (Lefranc, M.-P., TheImmunologist, 7, 132-136 (1999); Lefranc, M.-P. et al., Dev. Comp.Immunol., 27, 55-77 (2003) (“IMGT” numbering scheme).

For example, for classic formats, under Kabat, the CDR amino acidresidues in the heavy chain variable domain (VH) are numbered 31-35(HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acidresidues in the light chain variable domain (VL) are numbered 24-34(LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Under Chothia the CDR aminoacids in the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102(HCDR3); and the amino acid residues in VL are numbered 26-32 (LCDR1),50-52 (LCDR2), and 91-96 (LCDR3). By combining the CDR definitions ofboth Kabat and Chothia, the CDRs consist of amino acid residues 26-35(HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH and amino acidresidues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL.Under IMGT the CDR amino acid residues in the VH are numberedapproximately 26-35 (CDR1), 51-57 (CDR2) and 93-102 (CDR3), and the CDRamino acid residues in the VL are numbered approximately 27-32 (CDR1),50-52 (CDR2), and 89-97 (CDR3) (numbering according to “Kabat”). UnderIMGT, the CDR regions of an antibody can be determined using the programIMGT/DomainGap Align.

For example, under Kabat, the CDR amino acid residues of antibody NOV001in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1),50-65 (HCDR2), and 99-108 (HCDR3); and the CDR amino acid residues inthe light chain variable domain (VL) are numbered 24-39 (LCDR1), 55-61(LCDR2), and 94-102 (LCDR3). Under Chothia the CDR amino acids in the VHare numbered 26-32 (HCDR1), 52-57 (HCDR2), and 99-108 (HCDR3); and theamino acid residues in VL are numbered 26-39 (LCDR1), 55-57 (LCDR2), and96-101 (LCDR3). By combining the CDR definitions of both Kabat andChothia, the CDRs consist of amino acid residues 26-35 (HCDR1), 50-65(HCDR2), and 99-108 (HCDR3) in human VH and amino acid residues 24-39(LCDR1), 55-61 (LCDR2), and 94-102 (LCDR3) in human VL.

In another aspect, the present invention provides β-klotho bindingantibodies that comprise the heavy chain and light chain CDR1s, CDR2s,and CDR3s as described in Table 1, or combinations thereof. The aminoacid sequences of the VH CDR1s of the antibodies are shown in SEQ IDNOs: 3, 23, 43, and 63. The amino acid sequences of the VH CDR2s of theantibodies and are shown in SEQ ID NOs: 4, 24, 44, and 64. The aminoacid sequences of the VH CDR3s of the antibodies are shown in SEQ IDNOs: 5, 25, 45, and 65. The amino acid sequences of the VL CDR1s of theantibodies are shown in SEQ ID NOs: 13, 33, 53, and 73. The amino acidsequences of the VL CDR2s of the antibodies are shown in SEQ ID NOs: 14,34, 54, and 74. The amino acid sequences of the VL CDR3s of theantibodies are shown in SEQ ID NOs: 15, 35, 55, and 75. These CDRregions are delineated using the Kabat system.

Alternatively, as defined using the Chothia system (Al-Lazikani et al.,(1997) JMB 273, 927-948), the amino acid sequences of the VH CDR1s ofthe antibodies are shown in SEQ ID NOs: 6, 26, 46, and 66. The aminoacid sequences of the VH CDR2s of the antibodies and are shown in SEQ IDNOs: 7, 27, 47, and 67. The amino acid sequences of the VH CDR3s of theantibodies are shown in SEQ ID NOs: 8, 28, 48, and 68. The amino acidsequences of the VL CDR1s of the antibodies are shown in SEQ ID NOs: 16,36, 56, and 76. The amino acid sequences of the VL CDR2s of theantibodies are shown in SEQ ID NOs: 17, 37, 57, and 77. The amino acidsequences of the VL CDR3s of the antibodies are shown in SEQ ID NOs: 18,38, 58, and 78.

Alternatively, as defined using the Combined system, the amino acidsequences of the VH CDR1s of the antibodies are shown in SEQ ID NOs: 263and 268. The amino acid sequences of the VH CDR2s of the antibodies andare shown in SEQ ID NOs: 4, 24, 44, and 64. The amino acid sequences ofthe VH CDR3s of the antibodies are shown in SEQ ID NOs: 5, 25, and 45.The amino acid sequences of the VL CDR1s of the antibodies are shown inSEQ ID NOs: 13, 33, 53, and 73. The amino acid sequences of the VL CDR2sof the antibodies are shown in SEQ ID NOs: 14, 34, 54, and 74. The aminoacid sequences of the VL CDR3s of the antibodies are shown in SEQ IDNOs: 15, 35, 55, and 75.

Alternatively, as defined using the IMGT system, the amino acidsequences of the VH CDR1s of the antibodies are shown in SEQ ID NOs: 264and 269. The amino acid sequences of the VH CDR2s of the antibodies andare shown in SEQ ID NOs: 265 and 270. The amino acid sequences of the VHCDR3s of the antibodies are shown in SEQ ID NOs: 266 and 271. The aminoacid sequences of the VL CDR1s of the antibodies are shown in SEQ IDNOs: 267, and 272. The amino acid sequences of the VL CDR2s of theantibodies are shown in SEQ ID NOs: 17, 273, and 57. The amino acidsequences of the VL CDR3s of the antibodies are shown in SEQ ID NOs: 15,35, 55, and 75.

Given that each of these antibodies can bind to β-klotho and thatantigen-binding specificity is provided primarily by the CDR1, 2 and 3regions, the VH CDR1, 2 and 3 sequences and VL CDR1, 2 and 3 sequencescan be “mixed and matched” (i.e., CDRs from different antibodies can bemixed and matched, although each antibody preferably contains a VH CDR1,2 and 3 and a VL CDR1, 2 and 3 to create other β-klotho bindingmolecules of the invention. Such “mixed and matched” β-klotho bindingantibodies can be tested using the binding assays known in the art andthose described in the Examples (e.g., ELISAs, SET, Biacore® bindingassays). When VH CDR sequences are mixed and matched, the CDR1, CDR2and/or CDR3 sequence from a particular VH sequence should be replacedwith a structurally similar CDR sequence(s). Likewise, when VL CDRsequences are mixed and matched, the CDR1, CDR2 and/or CDR3 sequencefrom a particular VL sequence should be replaced with a structurallysimilar CDR sequence(s). It will be readily apparent to the ordinarilyskilled artisan that novel VH and VL sequences can be created bysubstituting one or more VH and/or VL CDR region sequences withstructurally similar sequences from the CDR sequences shown herein formonoclonal antibodies of the present invention. In addition to theforegoing, in one embodiment, the antigen-binding fragments of theantibodies described herein can comprise a VH CDR1, 2, and 3, or a VLCDR 1, 2, and 3, wherein the fragment binds to β-klotho as a singlevariable domain.

In certain embodiments of the invention, the antibodies orantigen-binding fragments thereof may have the heavy and light chainsequences of the Fabs described in Table 1. More specifically, theantibody or antigen-binding fragments thereof may have the heavy andlight sequence of Fab NOV001, NOV002, NOV003, NOV004.

In certain embodiments of the invention, the antibody or antigen-bindingfragment that specifically binds β-klotho comprises heavy chain variableregion CDR1, CDR2, and CDR3 of Fab NOV001, NOV002, NOV003, or NOV004,and light chain variable region CDR1, CDR2, and CDR3 of Fab NOV001,NOV002, NOV003, or NOV004, for example, as set forth in Table 1.

In other embodiments of the invention the antibody or antigen-bindingfragment in that specifically binds β-klotho comprises a heavy chainvariable region CDR1, a heavy chain variable region CDR2, a heavy chainvariable region CDR3, a light chain variable region CDR1, a light chainvariable region CDR2, and a light chain variable region CDR3 as definedby Kabat and described in Table 1. In still other embodiments of theinvention the antibody or antigen-binding fragment in that specificallybinds β-klotho comprises a heavy chain variable region CDR1, a heavychain variable region CDR2, a heavy chain variable region CDR3, a lightchain variable region CDR1, a light chain variable region CDR2, and alight chain variable region CDR3 as defined by Chothia and described inTable 1. In still other embodiments of the invention the antibody orantigen-binding fragment in that specifically binds β-klotho comprises aheavy chain variable region CDR1, a heavy chain variable region CDR2, aheavy chain variable region CDR3, a light chain variable region CDR1, alight chain variable region CDR2, and a light chain variable region CDR3as defined by Combined Kabat and Chothia and described in Table 1. Instill other embodiments of the invention the antibody or antigen-bindingfragment in that specifically binds β-klotho comprises a heavy chainvariable region CDR1, a heavy chain variable region CDR2, a heavy chainvariable region CDR3, a light chain variable region CDR1, a light chainvariable region CDR2, and a light chain variable region CDR3 as definedby IMGT and described in Table 1.

In a specific embodiment, the invention includes an antibody thatspecifically binds to β-klotho comprising a heavy chain variable regionCDR1 of SEQ ID NO: 3; a heavy chain variable region CDR2 of SEQ ID NO:4; a heavy chain variable region CDR3 of SEQ ID NO: 5; a light chainvariable region CDR1 of SEQ ID NO: 13; a light chain variable regionCDR2 of SEQ ID NO: 14; and a light chain variable region CDR3 of SEQ IDNO: 15.

In a specific embodiment, the invention includes an antibody thatspecifically binds to β-klotho comprising a heavy chain variable regionCDR1 of SEQ ID NO: 23; a heavy chain variable region CDR2 of SEQ ID NO:24; a heavy chain variable region CDR3 of SEQ ID NO: 25; a light chainvariable region CDR1 of SEQ ID NO: 33; a light chain variable regionCDR2 of SEQ ID NO: 34; and a light chain variable region CDR3 of SEQ IDNO: 35.

In a specific embodiment, the invention includes an antibody thatspecifically binds to β-klotho comprising a heavy chain variable regionCDR1 of SEQ ID NO: 43; a heavy chain variable region CDR2 of SEQ ID NO:44; a heavy chain variable region CDR3 of SEQ ID NO: 45; a light chainvariable region CDR1 of SEQ ID NO: 53; a light chain variable regionCDR2 of SEQ ID NO: 54; and a light chain variable region CDR3 of SEQ IDNO: 55.

In a specific embodiment, the invention includes an antibody thatspecifically binds to β-klotho comprising a heavy chain variable regionCDR1 of SEQ ID NO: 63; a heavy chain variable region CDR2 of SEQ ID NO:64; a heavy chain variable region CDR3 of SEQ ID NO: 65; a light chainvariable region CDR1 of SEQ ID NO: 73; a light chain variable regionCDR2 of SEQ ID NO: 74; and a light chain variable region CDR3 of SEQ IDNO: 75.

In a specific embodiment, the invention includes an antibody thatspecifically binds to β-klotho comprising a heavy chain variable regionCDR1 of SEQ ID NO: 6; a heavy chain variable region CDR2 of SEQ ID NO:7; a heavy chain variable region CDR3 of SEQ ID NO: 8; a light chainvariable region CDR1 of SEQ ID NO: 16; a light chain variable regionCDR2 of SEQ ID NO: 17; and a light chain variable region CDR3 of SEQ IDNO: 18.

In a specific embodiment, the invention includes an antibody thatspecifically binds to β-klotho comprising a heavy chain variable regionCDR1 of SEQ ID NO: 26; a heavy chain variable region CDR2 of SEQ ID NO:27; a heavy chain variable region CDR3 of SEQ ID NO: 28; a light chainvariable region CDR1 of SEQ ID NO: 36; a light chain variable regionCDR2 of SEQ ID NO: 37; and a light chain variable region CDR3 of SEQ IDNO: 38.

In a specific embodiment, the invention includes an antibody thatspecifically binds to β-klotho comprising a heavy chain variable regionCDR1 of SEQ ID NO: 46; a heavy chain variable region CDR2 of SEQ ID NO:47; a heavy chain variable region CDR3 of SEQ ID NO: 48; a light chainvariable region CDR1 of SEQ ID NO: 56; a light chain variable regionCDR2 of SEQ ID NO: 57; and a light chain variable region CDR3 of SEQ IDNO: 58.

In a specific embodiment, the invention includes an antibody thatspecifically binds to β-klotho comprising a heavy chain variable regionCDR1 of SEQ ID NO: 66; a heavy chain variable region CDR2 of SEQ ID NO:67; a heavy chain variable region CDR3 of SEQ ID NO: 68; a light chainvariable region CDR1 of SEQ ID NO: 76; a light chain variable regionCDR2 of SEQ ID NO: 77; and a light chain variable region CDR3 of SEQ IDNO: 78.

In a specific embodiment, the invention includes an antibody thatspecifically binds to β-klotho comprising a heavy chain variable regionCDR1 of SEQ ID NO: 263; a heavy chain variable region CDR2 of SEQ ID NO:4; a heavy chain variable region CDR3 of SEQ ID NO: 5; a light chainvariable region CDR1 of SEQ ID NO: 13; a light chain variable regionCDR2 of SEQ ID NO: 14; and a light chain variable region CDR3 of SEQ IDNO: 15.

In a specific embodiment, the invention includes an antibody thatspecifically binds to β-klotho comprising a heavy chain variable regionCDR1 of SEQ ID NO: 268; a heavy chain variable region CDR2 of SEQ ID NO:24; a heavy chain variable region CDR3 of SEQ ID NO: 25; a light chainvariable region CDR1 of SEQ ID NO: 33; a light chain variable regionCDR2 of SEQ ID NO: 34; and a light chain variable region CDR3 of SEQ IDNO: 35.

In a specific embodiment, the invention includes an antibody thatspecifically binds to β-klotho comprising a heavy chain variable regionCDR1 of SEQ ID NO: 263; a heavy chain variable region CDR2 of SEQ ID NO:44; a heavy chain variable region CDR3 of SEQ ID NO: 45; a light chainvariable region CDR1 of SEQ ID NO: 53; a light chain variable regionCDR2 of SEQ ID NO: 54; and a light chain variable region CDR3 of SEQ IDNO: 55.

In a specific embodiment, the invention includes an antibody thatspecifically binds to β-klotho comprising a heavy chain variable regionCDR1 of SEQ ID NO: 268; a heavy chain variable region CDR2 of SEQ ID NO:64; a heavy chain variable region CDR3 of SEQ ID NO: 25; a light chainvariable region CDR1 of SEQ ID NO: 73; a light chain variable regionCDR2 of SEQ ID NO: 74; and a light chain variable region CDR3 of SEQ IDNO: 75.

In a specific embodiment, the invention includes an antibody thatspecifically binds to β-klotho comprising a heavy chain variable regionCDR1 of SEQ ID NO: 264; a heavy chain variable region CDR2 of SEQ ID NO:265; a heavy chain variable region CDR3 of SEQ ID NO: 266; a light chainvariable region CDR1 of SEQ ID NO: 267; a light chain variable regionCDR2 of SEQ ID NO: 17; and a light chain variable region CDR3 of SEQ IDNO: 15.

In a specific embodiment, the invention includes an antibody thatspecifically binds to β-klotho comprising a heavy chain variable regionCDR1 of SEQ ID NO: 269; a heavy chain variable region CDR2 of SEQ ID NO:270; a heavy chain variable region CDR3 of SEQ ID NO: 271; a light chainvariable region CDR1 of SEQ ID NO: 272; a light chain variable regionCDR2 of SEQ ID NO: 273; and a light chain variable region CDR3 of SEQ IDNO: 35.

In a specific embodiment, the invention includes an antibody thatspecifically binds to β-klotho comprising a heavy chain variable regionCDR1 of SEQ ID NO: 264; a heavy chain variable region CDR2 of SEQ ID NO:265; a heavy chain variable region CDR3 of SEQ ID NO: 266; a light chainvariable region CDR1 of SEQ ID NO: 267; a light chain variable regionCDR2 of SEQ ID NO: 57; and a light chain variable region CDR3 of SEQ IDNO: 55.

In a specific embodiment, the invention includes an antibody thatspecifically binds to β-klotho comprising a heavy chain variable regionCDR1 of SEQ ID NO: 269; a heavy chain variable region CDR2 of SEQ ID NO:270; a heavy chain variable region CDR3 of SEQ ID NO: 271; a light chainvariable region CDR1 of SEQ ID NO: 272; a light chain variable regionCDR2 of SEQ ID NO: 273; and a light chain variable region CDR3 of SEQ IDNO: 75.

In certain embodiments, the invention includes antibodies orantigen-binding fragments that specifically bind to β-klotho asdescribed in Table 1. In a preferred embodiment, the antibody, orantigen-binding fragment, that binds β-klotho and activates the FGF21receptor complex is Fab NOV001, NOV002, NOV003, NOV004.

As used herein, a human antibody comprises heavy or light chain variableregions or full length heavy or light chains that are “the product of”or “derived from” a particular germline sequence if the variable regionsor full length chains of the antibody are obtained from a system thatuses human germline immunoglobulin genes. Such systems includeimmunizing a transgenic mouse carrying human immunoglobulin genes withthe antigen of interest or screening a human immunoglobulin gene librarydisplayed on phage with the antigen of interest. A human antibody thatis “the product of” or “derived from” a human germline immunoglobulinsequence can be identified as such by comparing the amino acid sequenceof the human antibody to the amino acid sequences of human germlineimmunoglobulins and selecting the human germline immunoglobulin sequencethat is closest in sequence (i.e., greatest % identity) to the sequenceof the human antibody.

A human antibody that is “the product of” or “derived from” a particularhuman germline immunoglobulin sequence may contain amino aciddifferences as compared to the germline sequence, due to, for example,naturally occurring somatic mutations or intentional introduction ofsite-directed mutations. However, in the VH or VL framework regions, aselected human antibody typically is at least 90% identical in aminoacids sequence to an amino acid sequence encoded by a human germlineimmunoglobulin gene and contains amino acid residues that identify thehuman antibody as being human when compared to the germlineimmunoglobulin amino acid sequences of other species (e.g., murinegermline sequences). In certain cases, a human antibody may be at least60%, 70%, 80%, 90%, or at least 95%, or even at least 96%, 97%, 98%, or99% identical in amino acid sequence to the amino acid sequence encodedby the germline immunoglobulin gene.

Typically, a recombinant human antibody will display no more than 10amino acid differences from the amino acid sequence encoded by the humangermline immunoglobulin gene in the VH or VL framework regions. Incertain cases, the human antibody may display no more than 5, or even nomore than 4, 3, 2, or 1 amino acid difference from the amino acidsequence encoded by the germline immunoglobulin gene. Examples of humangermline immunoglobulin genes include, but are not limited to thevariable domain germline fragments described below, as well as DP47 andDPK9.

Homologous Antibodies

In yet another embodiment, the present invention provides an antibody,or an antigen-binding fragment thereof, comprising amino acid sequencesthat are homologous to the sequences described in Table 1, and theantibody binds to a β-klotho protein (e.g., human and cynomolgus monkeyβ-klotho), and retains the desired functional properties of thoseantibodies described in Table 1.

For example, the invention provides an isolated antibody, or afunctional antigen-binding fragment thereof, comprising a heavy chainvariable domain and a light chain variable domain, wherein the heavychain variable domain comprises an amino acid sequence that is at least80%, at least 90%, 95%, 96%, 97%, 98%, or at least 99% identical to anamino acid sequence selected from the group consisting of SEQ ID NOs: 9,29, 49, or 69; the light chain variable domain comprises an amino acidsequence that is at least 80%, at least 90%, 95%, 96%, 97%, 98%, or atleast 99% identical to an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 19, 39, 59, or 79; and the antibodyspecifically binds to β-klotho (e.g., human and cynomolgus monkeyβ-klotho). In certain aspects of the invention the heavy and light chainsequences further comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3sequences as defined by Kabat, for example SEQ ID NOs: 3, 4, 5, 13, 14,and 15, respectively. In certain other aspects of the invention theheavy and light chain sequences further comprise HCDR1, HCDR2, HCDR3,LCDR1, LCDR2, and LCDR3 sequences as defined by Chothia, for example SEQID NOs: 6, 7, 8, 16, 17, and 18, respectively. In certain other aspectsof the invention the heavy and light chain sequences further compriseHCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences as defined byCombined, for example SEQ ID NOs: 263, 4, 5, 13, 14, and 15,respectively. In certain other aspects of the invention the heavy andlight chain sequences further comprise HCDR1, HCDR2, HCDR3, LCDR1,LCDR2, and LCDR3 sequences as defined by IMGT, for example SEQ ID NOs:264, 265, 266, 267, 17, and 15, respectively.

For example, the invention provides an isolated antibody, or afunctional antigen-binding fragment thereof, comprising a heavy chainvariable domain and a light chain variable domain, wherein the heavychain variable domain comprises an amino acid sequence that is at least80%, at least 90%, 95%, 96%, 97%, 98%, or at least 99% identical to anamino acid sequence of SEQ ID NOs: 9; the light chain variable domaincomprises an amino acid sequence that is at least 80%, at least 90%,95%, 96%, 97%, 98%, or at least 99% identical to an amino acid sequenceof SEQ ID NOs: 19; and the antibody specifically binds to β-klotho(e.g., human and cynomolgus monkey β-klotho). In certain aspects of theinvention the heavy and light chain sequences further comprise HCDR1,HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences as defined by Kabat, forexample SEQ ID NOs: 3, 4, 5, 13, 14, and 15, respectively. In certainother aspects of the invention the heavy and light chain sequencesfurther comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequencesas defined by Chothia, for example SEQ ID NOs: 6, 7, 8, 16, 17, and 18,respectively. In certain other aspects of the invention the heavy andlight chain sequences further comprise HCDR1, HCDR2, HCDR3, LCDR1,LCDR2, and LCDR3 sequences as defined by Combined, for example SEQ IDNOs: 263, 4, 5, 13, 14, and 15, respectively. In certain other aspectsof the invention the heavy and light chain sequences further compriseHCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences as defined byIMGT, for example SEQ ID NOs: 264, 265, 266, 267, 17, and 15,respectively.

For example, the invention provides an isolated antibody, or afunctional antigen-binding fragment thereof, comprising a heavy chainvariable domain and a light chain variable domain, wherein the heavychain variable domain comprises an amino acid sequence that is at least80%, at least 90%, 95%, 96%, 97%, 98%, or at least 99% identical to anamino acid sequence of SEQ ID NOs: 29; the light chain variable domaincomprises an amino acid sequence that is at least 80%, at least 90%,95%, 96%, 97%, 98%, or at least 99% identical to an amino acid sequenceof SEQ ID NO: 39; and the antibody specifically binds to β-klotho (e.g.,human and cynomolgus monkey β-klotho). In certain aspects of theinvention the heavy and light chain sequences further comprise HCDR1,HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences as defined by Kabat, forexample SEQ ID NOs: 23, 24, 25, 33, 34, and 35, respectively. In certainother aspects of the invention the heavy and light chain sequencesfurther comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequencesas defined by Chothia, for example SEQ ID NOs: 26, 27, 28, 36, 37, and38, respectively. In certain other aspects of the invention the heavyand light chain sequences further comprise HCDR1, HCDR2, HCDR3, LCDR1,LCDR2, and LCDR3 sequences as defined by Combined, for example SEQ IDNOs: 268, 24, 25, 33, 34, and 35, respectively. In certain other aspectsof the invention the heavy and light chain sequences further compriseHCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences as defined byIMGT, for example SEQ ID NOs: 269, 270, 271, 272, 273, and 35,respectively.

For example, the invention provides an isolated antibody, or afunctional antigen-binding fragment thereof, comprising a heavy chainvariable domain and a light chain variable domain, wherein the heavychain variable domain comprises an amino acid sequence that is at least80%, at least 90%, 95%, 96%, 97%, 98%, or at least 99% identical to anamino acid sequence of SEQ ID NOs: 49; the light chain variable domaincomprises an amino acid sequence that is at least 80%, at least 90%,95%, 96%, 97%, 98%, or at least 99% identical to an amino acid sequenceof SEQ ID NOs: 59; and the antibody specifically binds to β-klotho(e.g., human and cynomolgus monkey β-klotho). In certain aspects of theinvention the heavy and light chain sequences further comprise HCDR1,HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences as defined by Kabat, forexample SEQ ID NOs: 43, 44, 45, 53, 54, and 55, respectively. In certainother aspects of the invention the heavy and light chain sequencesfurther comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequencesas defined by Chothia, for example SEQ ID NOs: 46, 47, 48, 56, 57, and58, respectively. In certain other aspects of the invention the heavyand light chain sequences further comprise HCDR1, HCDR2, HCDR3, LCDR1,LCDR2, and LCDR3 sequences as defined by Combined, for example SEQ IDNOs: 263, 44, 45, 53, 54, and 55, respectively. In certain other aspectsof the invention the heavy and light chain sequences further compriseHCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences as defined byIMGT, for example SEQ ID NOs: 264, 265, 266, 267, 57, and 55,respectively.

For example, the invention provides an isolated antibody, or afunctional antigen-binding fragment thereof, comprising a heavy chainvariable domain and a light chain variable domain, wherein the heavychain variable domain comprises an amino acid sequence that is at least80%, at least 90%, 95%, 96%, 97%, 98%, or at least 99% identical to anamino acid sequence of SEQ ID NOs: 69; the light chain variable domaincomprises an amino acid sequence that is at least 80%, at least 90%,95%, 96%, 97%, 98%, or at least 99% identical to an amino acid sequenceof SEQ ID NOs: 79; and the antibody specifically binds to β-klotho(e.g., human and cynomolgus monkey β-klotho). In certain aspects of theinvention the heavy and light chain sequences further comprise HCDR1,HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences as defined by Kabat, forexample SEQ ID NOs: 63, 64, 65, 73, 74, and 75, respectively. In certainother aspects of the invention the heavy and light chain sequencesfurther comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequencesas defined by Chothia, for example SEQ ID NOs: 66, 67, 68, 76, 77, and78, respectively. In certain other aspects of the invention the heavyand light chain sequences further comprise HCDR1, HCDR2, HCDR3, LCDR1,LCDR2, and LCDR3 sequences as defined by Combined, for example SEQ IDNOs: 268, 64, 25, 73, 74, and 75, respectively. In certain other aspectsof the invention the heavy and light chain sequences further compriseHCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences as defined byIMGT, for example SEQ ID NOs: 269, 270, 271, 272, 273, and 75,respectively.

In other embodiments, the VH and/or VL amino acid sequences may be 50%,60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the sequencesset forth in Table 1. In other embodiments, the VH and/or VL amino acidsequences may be identical except for an amino acid substitution in nomore than 1, 2, 3, 4 or 5 amino acid positions. An antibody having VHand VL regions having high (i.e., 80% or greater) identity to the VH andVL regions of those described in Table 1 can be obtained by mutagenesis(e.g., site-directed or PCR-mediated mutagenesis) of nucleic acidmolecules encoding SEQ ID NOs: 10, 30, 50, or 70 and SEQ ID NOs: 20, 40,60, or 80, respectively, followed by testing of the encoded alteredantibody for retained function using the functional assays describedherein.

In other embodiments, the full length heavy chain and/or full lengthlight chain amino acid sequences may be 50% 60%, 70%, 80%, 90%, 95%,96%, 97%, 98% or 99% identical to the sequences set forth in Table 1. Anantibody having a full length heavy chain and full length light chainhaving high (i.e., 80% or greater) identity to the full length heavychains of any of SEQ ID NOs: 9, 29, 49, or 69, and full length lightchains of any of SEQ ID NOs: 19, 39, 59, or 79, can be obtained bymutagenesis (e.g., site-directed or PCR-mediated mutagenesis) of nucleicacid molecules encoding such polypeptides, followed by testing of theencoded altered antibody for retained function using the functionalassays described herein. An antibody having a full length heavy chainand full length light chain having high (i.e., 80% or greater) identityto the full length heavy chains of any of SEQ ID NOs: 11, 31, 51, or 71,and full length light chains of any of SEQ ID NOs: 21, 41, 61, or 81,can be obtained by mutagenesis (e.g., site-directed or PCR-mediatedmutagenesis) of nucleic acid molecules encoding such polypeptides,followed by testing of the encoded altered antibody for retainedfunction using the functional assays described herein.

In other embodiments, the full length heavy chain and/or full lengthlight chain nucleotide sequences may be 60%, 70%, 80%, 90%, 95%, 96%,97%, 98% or 99% identical to the sequences set forth in Table 1.

In other embodiments, the variable regions of heavy chain and/or thevariable regions of light chain nucleotide sequences may be 60%, 70%,80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the sequences set forthin Table 1.

In some embodiments, the heavy chain and/or the light chain CDRs may be90%, 95%, 96%, 97%, 98% or 99% identical to the sequences set forth inTable 1.

As used herein, the percent identity between the two sequences is afunction of the number of identical positions shared by the sequences(i.e., % identity equals number of identical positions/total number ofpositions×100), taking into account the number of gaps, and the lengthof each gap, which need to be introduced for optimal alignment of thetwo sequences. The comparison of sequences and determination of percentidentity between two sequences can be accomplished using a mathematicalalgorithm, as described in the non-limiting examples below.

Additionally or alternatively, the protein sequences of the presentinvention can further be used as a “query sequence” to perform a searchagainst public databases to, for example, identify related sequences.For example, such searches can be performed using the BLAST program(version 2.0) of Altschul, et al., 1990 J. Mol. Biol. 215:403-10.

Antibodies with Conservative Modifications

In certain embodiments, an antibody of the invention has a heavy chainvariable region comprising CDR1, CDR2, and CDR3 sequences and a lightchain variable region comprising CDR1, CDR2, and CDR3 sequences, whereinone or more of these CDR sequences have specified amino acid sequencesbased on the antibodies described herein or conservative modificationsthereof, and wherein the antibodies retain the desired functionalproperties of the β-klotho-binding antibodies of the invention.

Accordingly, the invention provides an isolated antibody, or aantigen-binding fragment thereof, consisting of a heavy chain variableregion comprising CDR1, CDR2, and CDR3 sequences and a light chainvariable region comprising CDR1, CDR2, and CDR3 sequences, wherein: theheavy chain variable region CDR1 amino acid sequences are selected fromthe group consisting of SEQ ID NOs: 3, 23, 43, and 63, and conservativemodifications thereof; the heavy chain variable region CDR2 amino acidsequences are selected from the group consisting of SEQ ID NOs: 4, 24,44, and 64, and conservative modifications thereof, the heavy chainvariable region CDR3 amino acid sequences are selected from the groupconsisting of SEQ ID NOs: 5, 25, 45, and 65, and conservativemodifications thereof; the light chain variable regions CDR1 amino acidsequences are selected from the group consisting of SEQ ID NOs: 13, 33,53, and 73, and conservative modifications thereof, the light chainvariable regions CDR2 amino acid sequences are selected from the groupconsisting of SEQ ID NOs: 14, 34, 54, and 74, and conservativemodifications thereof; the light chain variable regions of CDR3 aminoacid sequences are selected from the group consisting of SEQ ID NOs: 15,35, 55, and 75, and conservative modifications thereof; and the antibodyor antigen-binding fragments thereof specifically binds to β-klotho.

Accordingly, the invention provides an isolated antibody, or aantigen-binding fragment thereof, consisting of a heavy chain variableregion comprising CDR1, CDR2, and CDR3 sequences and a light chainvariable region comprising CDR1, CDR2, and CDR3 sequences, wherein: theheavy chain variable region CDR1 amino acid sequences are selected fromthe group consisting of SEQ ID NOs: 263 and 268, and conservativemodifications thereof; the heavy chain variable region CDR2 amino acidsequences are selected from the group consisting of SEQ ID NOs: 4, 24,44, and 64, and conservative modifications thereof, the heavy chainvariable region CDR3 amino acid sequences are selected from the groupconsisting of SEQ ID NOs: 5, 25, and 45, and conservative modificationsthereof; the light chain variable regions CDR1 amino acid sequences areselected from the group consisting of SEQ ID NOs: 13, 33, 53, and 73,and conservative modifications thereof, the light chain variable regionsCDR2 amino acid sequences are selected from the group consisting of SEQID NOs: 14, 34, 54, and 74, and conservative modifications thereof; thelight chain variable regions of CDR3 amino acid sequences are selectedfrom the group consisting of SEQ ID NOs: 15, 35, 55, and 75, andconservative modifications thereof; and the antibody or antigen-bindingfragments thereof specifically binds to β-klotho.

In other embodiments, the antibody of the invention is optimized forexpression in a mammalian cell has a full length heavy chain sequenceand a full length light chain sequence, wherein one or more of thesesequences have specified amino acid sequences based on the antibodiesdescribed herein or conservative modifications thereof, and wherein theantibodies retain the desired functional properties of the β-klothobinding antibodies of the invention. Accordingly, the invention providesan isolated antibody optimized for expression in a mammalian cellconsisting of a full length heavy chain and a full length light chainwherein the full length heavy chain has amino acid sequences selectedfrom the group of SEQ ID NOs: 11, 31, 51, or 71, and conservativemodifications thereof; and the full length light chain has amino acidsequences selected from the group of SEQ ID NOs: 21, 41, 61, or 81, andconservative modifications thereof, and the antibody specifically bindsto β-klotho (e.g., human and cynomolgus monkey β-klotho).

Antibodies that Bind to the Same Epitope

The present invention provides antibodies that bind to the same epitopeas the β-klotho binding antibodies described in Table 1 (e.g., NOV001,NOV002, NOV003, or NOV004). In a particular aspect, such antibodies andantigen-binding fragments are capable of increasing the activity ofβ-klotho and FGFR1c. Additional antibodies can therefore be identifiedbased on their ability to compete (e.g., to competitively inhibit thebinding of, in a statistically significant manner) with other antibodiesof the invention in β-klotho binding assays (such as those described inthe Examples). The ability of a test antibody to inhibit the binding ofantibodies of the present invention to a β-klotho protein demonstratesthat the test antibody can compete with that antibody for binding toβ-klotho; such an antibody may, according to non-limiting theory, bindto the same or a related (e.g., a structurally similar or spatiallyproximal) epitope on the β-klotho protein as the antibody with which itcompetes. In a certain embodiment, the antibody that binds to the sameepitope on β-klotho as the antibodies of the present invention is ahuman monoclonal antibody. Such human monoclonal antibodies can beprepared and isolated as described herein. As used herein, an antibody“competes” for binding when the competing antibody inhibits β-klothobinding of an antibody or antigen-binding fragment of the invention bymore than 50% (for example, 80%, 85%, 90%, 95%, 98% or 99%) in thepresence of an equimolar concentration of competing antibody. In acertain embodiment, the antibody that binds to the same epitope onβ-klotho as the antibodies of the present invention is a humanizedmonoclonal antibody. Such humanized monoclonal antibodies can beprepared and isolated as described herein.

In a particular aspect, the present invention provides antibodies thatbind to the same epitope as β-klotho binding antibody NOV001. In aparticular aspect, the present invention provides antibodies that bindto the same epitope as β-klotho binding antibody NOV002. In a particularaspect, the present invention provides antibodies that bind to the sameepitope as (3-klotho binding antibody NOV003. In a particular aspect,the present invention provides antibodies that bind to the same epitopeas β-klotho binding antibody NOV004.

In a particular aspect, the present invention provides antibodies thatbind to the same epitope as a β-klotho binding antibody which comprisesa heavy chain variable region (VH) comprising the amino acid sequence ofSEQ ID NO: 9 and a light chain variable region (VL) comprising the aminoacid sequence of SEQ ID NO: 19.

In a particular aspect, the present invention provides antibodies thatbind to the same epitope as a β-klotho binding antibody which comprisesa heavy chain variable region (VH) comprising the amino acid sequence ofSEQ ID NO: 29 and a light chain variable region (VL) comprising theamino acid sequence of SEQ ID NO: 39.

In a particular aspect, the present invention provides antibodies thatbind to the same epitope as a β-klotho binding antibody which comprisesa heavy chain variable region (VH) comprising the amino acid sequence ofSEQ ID NO: 49 and a light chain variable region (VL) comprising theamino acid sequence of SEQ ID NO: 59.

In a particular aspect, the present invention provides antibodies thatbind to the same epitope as a β-klotho binding antibody which comprisesa heavy chain variable region (VH) comprising the amino acid sequence ofSEQ ID NO: 69 and a light chain variable region (VL) comprising theamino acid sequence of SEQ ID NO: 79.

In specific aspects, provided herein are isolated antibodies or anantigen-binding fragment thereof that bind to an epitope of β-klotho,wherein the epitope comprises one or more of the SEQ ID NOs shown inTable 2. In a particular aspect, such antibodies and antigen-bindingfragments are capable of increasing the activity of β-klotho and FGFR1c.

In a particular aspect, provided herein is an isolated antibody orantigen-binding fragment thereof that binds to an epitope of β-klotho,wherein said epitope comprises one or more amino acids of residues246-265 of the β-klotho sequence (SEQ ID NO:262). In a particularaspect, provided herein is an isolated antibody or antigen-bindingfragment thereof that binds to an epitope of β-klotho, wherein saidepitope comprises one or more amino acids of residues 536-550 of theβ-klotho sequence (SEQ ID NO:262). In a particular aspect, providedherein is an isolated antibody or antigen-binding fragment thereof thatbinds to an epitope of β-klotho, wherein said epitope comprises one ormore amino acids of residues 834-857 of the β-klotho sequence (SEQ IDNO:262). In a particular aspect, provided herein is an isolated antibodyor antigen-binding fragment thereof that binds to an epitope ofβ-klotho, wherein said epitope comprises one or more amino acids ofresidues 959-986 of the β-klotho sequence (SEQ ID NO:262).

In a particular aspect, provided herein is an isolated antibody orantigen-binding fragment thereof that binds to an epitope of β-klotho,wherein said epitope comprises one or more amino acids of residues246-265, 536-550, 834-857 and 959-986 of the β-klotho sequence (SEQ IDNO:262). In specific aspects, provided herein is an isolated antibody orantigen-binding fragment thereof that binds to an epitope of β-klotho,wherein said epitope comprises two or more amino acids of residues246-265, 536-550, 834-857 and 959-986 of the β-klotho sequence (SEQ IDNO:262). In specific aspects, provided herein is an isolated antibody orantigen-binding fragment thereof that binds to an epitope of β-klotho,wherein said epitope comprises three or more amino acids of residues246-265, 536-550, 834-857 and 959-986 of the β-klotho sequence (SEQ IDNO:262). In specific aspects, provided herein is an isolated antibody orantigen-binding fragment thereof that binds to an epitope of β-klotho,wherein said epitope comprises amino acids of residues 246-265, 536-550,834-857 and 959-986 of the β-klotho sequence (SEQ ID NO:262).

In a particular aspect, provided herein is an isolated antibody orantigen-binding fragment thereof that binds to one or more epitopes ofβ-klotho, wherein said epitopes comprises one or more of amino acids ofresidues 646-670 of the β-klotho sequence (SEQ ID NO:262). In aparticular aspect, provided herein is an isolated antibody orantigen-binding fragment thereof that binds to one or more epitopes ofβ-klotho, wherein said epitopes comprises one or more of amino acids ofresidues 696-700 of the β-klotho sequence (SEQ ID NO:262). In aparticular aspect, provided herein is an isolated antibody orantigen-binding fragment thereof that binds to one or more epitopes ofβ-klotho, wherein said epitopes comprises one or more of amino acids ofresidues 646-689 of the β-klotho sequence (SEQ ID NO:262).

In a particular aspect, provided herein is an isolated antibody orantigen-binding fragment thereof that binds to one or more epitopes ofβ-klotho, wherein said epitopes comprises one, or two, or three, orfour, or five, or more of amino acids of residues 646-670, 696-700, and646-689 of the β-klotho sequence (SEQ ID NO:262). In a certain aspect,provided herein is an isolated antibody or antigen-binding fragmentthereof that binds to two or more epitopes of β-klotho, wherein saidepitopes comprises one or more of amino acids of residues 646-670,696-700, and 646-689 of the β-klotho sequence (SEQ ID NO:262). In aspecific aspect, provided herein is an isolated antibody orantigen-binding fragment thereof that binds to three or more epitopes ofβ-klotho, wherein said epitopes comprises one or more of amino acids ofresidues 646-670, 696-700, and 646-689 of the β-klotho sequence (SEQ IDNO:262). In a specific aspect, provided herein is an isolated antibodyor antigen-binding fragment thereof that binds to three or more epitopesof β-klotho, wherein said epitopes comprises amino acids of residues646-670, 696-700, and 646-689 of the β-klotho sequence (SEQ ID NO:262).

In specific aspects, provided herein are isolated antibodies or anantigen-binding fragment thereof that bind to an epitope of β-klotho,wherein the epitope comprises one or more of the SEQ ID NOs shown inTable 2, wherein said antibodies and antigen-binding fragments arecapable of increasing the activity of β-klotho and FGFR1c, and whereinsaid antibodies and antigen-binding fragments are capable of protecting,by hydrogen-deuterium exchange (HDx), one or more peptides of β-klothoas characterized by a change in deuterium incorporation in the range of−0.5 to −2.1, for example as set forth in Table 2.

In a particular aspect, provided herein is an isolated antibody orantigen-binding fragment thereof that protects, as determined byhydrogen-deuterium exchange (HDx), one, two, three, four, five, or moreof the following peptides of β-klotho (SEQ ID NO: 262): 245-266,246-265, 343-349, 344-349, 421-429, 488-498, 509-524, 536-550, 568-576,646-669, 646-670, 696-700, 773-804, 834-857, and 959-986 aa.

In a specific aspect, provided herein is an isolated antibody orantigen-binding fragment thereof, which increases the activity ofβ-klotho and FGFR1c, wherein the antibody or antigen-binding fragmentthereof protects, as determined by hydrogen-deuterium exchange (HDx),one, two, three, four, five, or more peptides from the following as setforth in Table 2: SEQ ID NOs: 109, 110, 111, 112, 113, 125, 126, 127,128, 129, 141, 142, 143, 156, 157, 158, 159, 160, 161, 163, 164, 165,167, 168, 169, 170, 171, 172, 184, 185, 186, 187, 188, 195, 196, 197,198, 204, 212, 213, 214, 215, 216, 217, 224, 225, 226, 227, 228, 229,230, 231, 232, 233, 256, 257, 258, 259, 260, and 261.

In certain aspects, provided herein is isolated antibody orantigen-binding fragment thereof, which increases the activity ofβ-klotho and FGFR1c, wherein the antibody or antigen-binding fragmentthereof protects, as determined by hydrogen-deuterium exchange (HDx),six, seven, eight, nine, ten, or more peptides from the following as setforth in Table 2: SEQ ID NOs: 109, 110, 111, 112, 113, 125, 126, 127,128, 129, 141, 142, 143, 156, 157, 158, 159, 160, 161, 163, 164, 165,167, 168, 169, 170, 171, 172, 184, 185, 186, 187, 188, 195, 196, 197,198, 204, 212, 213, 214, 215, 216, 217, 224, 225, 226, 227, 228, 229,230, 231, 232, 233, 256, 257, 258, 259, 260, and 261.

In certain aspects, provided herein is isolated antibody orantigen-binding fragment thereof, which increases the activity ofβ-klotho and FGFR1c, wherein the antibody or antigen-binding fragmentthereof does not contact residues 701 (Tyr) or 703 (Arg) of humanβ-klotho (SEQ ID NO: 262).Engineered and Modified Antibodies

An antibody of the invention further can be prepared using an antibodyhaving one or more of the VH and/or VL sequences shown herein asstarting material to engineer a modified antibody, which modifiedantibody may have altered properties from the starting antibody. Anantibody can be engineered by modifying one or more residues within oneor both variable regions (i.e., VH and/or VL), for example within one ormore CDR regions and/or within one or more framework regions.Additionally or alternatively, an antibody can be engineered bymodifying residues within the constant region(s), for example to alterthe effector function(s) of the antibody.

One type of variable region engineering that can be performed is CDRgrafting. Antibodies interact with target antigens predominantly throughamino acid residues that are located in the six heavy and light chaincomplementarity determining regions (CDRs). For this reason, the aminoacid sequences within CDRs are more diverse between individualantibodies than sequences outside of CDRs. Because CDR sequences areresponsible for most antibody-antigen interactions, it is possible toexpress recombinant antibodies that mimic the properties of specificnaturally occurring antibodies by constructing expression vectors thatinclude CDR sequences from the specific naturally occurring antibodygrafted onto framework sequences from a different antibody withdifferent properties (see, e.g., Riechmann, L. et al., 1998 Nature332:323-327; Jones, P. et al., 1986 Nature 321:522-525; Queen, C. etal., 1989 Proc. Natl. Acad., U.S.A. 86:10029-10033; U.S. Pat. No.5,225,539 to Winter, and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762and 6,180,370 to Queen et al.)

Accordingly, another embodiment of the invention pertains to an isolatedantibody, or an antigen-binding fragment thereof, comprising a heavychain variable region comprising CDR1 sequences having an amino acidsequence selected from the HCDR1 sequences set forth in Table 1; CDR2sequences having an amino acid sequence selected from the HCDR2sequences set forth in Table 1; CDR3 sequences having an amino acidsequence selected from the HCDR3 sequences set forth in table 1; and alight chain variable region having CDR1 sequences having an amino acidsequence selected from the LCDR1 sequences set forth in Table 1; CDR2sequences having an amino acid sequence selected from the LCDR2sequences set forth in Table 1; and CDR3 sequences consisting of anamino acid sequence selected from the LCDR3 sequences set forth inTable 1. Thus, such antibodies contain the VH and VL CDR sequences ofmonoclonal antibodies, yet may contain different framework sequencesfrom these antibodies.

Accordingly, another embodiment of the invention pertains to an isolatedantibody, or an antigen-binding fragment thereof, comprising a heavychain variable region comprising CDR1 sequences having an amino acidsequence selected from the group consisting of SEQ ID NOs: 3, 23, 43,and 63; CDR2 sequences having an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 4, 24, 44, and 64; CDR3 sequences havingan amino acid sequence selected from the group consisting of SEQ ID NOs:5, 25, 45, and 65, respectively; and a light chain variable regionhaving CDR1 sequences having an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 13, 33, 53, and 73; CDR2 sequenceshaving an amino acid sequence selected from the group consisting of SEQID NOs: 14, 34, 54, and 74; and CDR3 sequences consisting of an aminoacid sequence selected from the group consisting of SEQ ID NOs: 15, 35,55, and 75, respectively. Thus, such antibodies contain the VH and VLCDR sequences of monoclonal antibodies, yet may contain differentframework sequences from these antibodies.

Such framework sequences can be obtained from public DNA databases orpublished references that include germline antibody gene sequences. Forexample, germline DNA sequences for human heavy and light chain variableregion genes can be found in the “VBase” human germline sequencedatabase (available on the world wide web at mrc-cpe.cam.ac.uk/vbase),as well as in Kabat, E. A., et al., 1991 Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242; Tomlinson, I. M., et al.,1992 J. Mol. Biol. 227:776-798; and Cox, J. P. L. et al., 1994 Eur. JImmunol. 24:827-836; the contents of each of which are expresslyincorporated herein by reference.

An example of framework sequences for use in the antibodies of theinvention are those that are structurally similar to the frameworksequences used by selected antibodies of the invention, e.g., consensussequences and/or framework sequences used by monoclonal antibodies ofthe invention. The VH CDR1, 2 and 3 sequences, and the VL CDR1, 2 and 3sequences, can be grafted onto framework regions that have the identicalsequence as that found in the germline immunoglobulin gene from whichthe framework sequence derive, or the CDR sequences can be grafted ontoframework regions that contain one or more mutations as compared to thegermline sequences. For example, it has been found that in certaininstances it is beneficial to mutate residues within the frameworkregions to maintain or enhance the antigen-binding ability of theantibody (see e.g., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and6,180,370 to Queen et al). Frameworks that can be utilized as scaffoldson which to build the antibodies and antigen-binding fragments describedherein include, but are not limited to VH1A, VH1B, VH3, Vk1, Vl2, andVk2. Additional frameworks are known in the art and may be found, forexample, in the vBase data base on the world wide web atvbase.mrc-cpe.cam.ac.uk/index.php?&MMN_position=1:1.

Accordingly, an embodiment of the invention relates to isolated β-klothobinding antibodies, or antigen-binding fragments thereof, comprising aheavy chain variable region comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 9, 29, 49, or 69, or an aminoacid sequence having one, two, three, four or five amino acidsubstitutions, deletions or additions in the framework region of suchsequences, and further comprising a light chain variable region havingan amino acid sequence selected from the group consisting of SEQ ID NOs:19, 39, 59, or 79, or an amino acid sequence having one, two, three,four or five amino acid substitutions, deletions or additions in theframework region of such sequences.

Accordingly, an embodiment of the invention relates to isolated β-klothobinding antibodies, or antigen-binding fragments thereof, comprising aheavy chain variable region comprising the amino acid sequence of SEQ IDNO: 9, or an amino acid sequence having one, two, three, four or fiveamino acid substitutions, deletions or additions in the framework regionof such sequences, and further comprising a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 19, or an amino acidsequence having one, two, three, four or five amino acid substitutions,deletions or additions in the framework region of such sequences.

In a particular aspect, an embodiment of the invention relates toisolated β-klotho binding antibodies, or antigen-binding fragmentsthereof, comprising a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 29, or an amino acid sequence having one,two, three, four or five amino acid substitutions, deletions oradditions in the framework region of such sequences, and furthercomprising a light chain variable region comprising the amino acidsequence of SEQ ID NO: 39, or an amino acid sequence having one, two,three, four or five amino acid substitutions, deletions or additions inthe framework region of such sequences.

In a particular aspect, an embodiment of the invention relates toisolated β-klotho binding antibodies, or antigen-binding fragmentsthereof, comprising a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 49, or an amino acid sequence having one,two, three, four or five amino acid substitutions, deletions oradditions in the framework region of such sequences, and furthercomprising a light chain variable region comprising the amino acidsequence of SEQ ID NO: 59, or an amino acid sequence having one, two,three, four or five amino acid substitutions, deletions or additions inthe framework region of such sequences.

In a particular aspect, an embodiment of the invention relates toisolated β-klotho binding antibodies, or antigen-binding fragmentsthereof, comprising a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 69, or an amino acid sequence having one,two, three, four or five amino acid substitutions, deletions oradditions in the framework region of such sequences, and furthercomprising a light chain variable region comprising the amino acidsequence of SEQ ID NO: 79, or an amino acid sequence having one, two,three, four or five amino acid substitutions, deletions or additions inthe framework region of such sequences.

Another type of variable region modification is to mutate amino acidresidues within the VH and/or VL CDR1, CDR2 and/or CDR3 regions tothereby improve one or more binding properties (e.g., affinity) of theantibody of interest, known as “affinity maturation.” Site-directedmutagenesis or PCR-mediated mutagenesis can be performed to introducethe mutation(s) and the effect on antibody binding, or other functionalproperty of interest, can be evaluated in in vitro or in vivo assays asdescribed herein and provided in the Examples. Conservativemodifications (as discussed above) can be introduced. The mutations maybe amino acid substitutions, additions or deletions. Moreover, typicallyno more than one, two, three, four or five residues within a CDR regionare altered.

Certain amino acid sequence motifs are known to undergopost-translational modification (PTM) such as glycosylation (i.e. NxS/T,x any but P), oxidation of free cysteines, deamidation (e.g. NG) orisomerization (e.g. DG). If present in the CDR regions, those motifs areideally removed by site-directed mutagenesis in order to increaseproduct homogeneity.

The process of affinity maturation is well described in the art. Amongmany display systems, phage display (Smith G P (1985) Science228:1315-1317) and display on eukaryotic cells such as yeast (Boder E Tand Wittrup K D (1997) Nature Biotechnology 15: 553-557) seem to be themost commonly applied systems to select for antibody-antigeninteraction. Advantages of those display systems are that they aresuitable for a wide range of antigens and that the selection stringencycan be easily adjusted. In phage display, scFv or Fab fragments can bedisplayed and in yeast display full-length IgG in addition. Thosecommonly applied methods allow selection of a desired antibody variantsfrom larger libraries with diversities of more than 10E7. Libraries withsmaller diversity, e.g. 10E3, may be screen by micro-expression andELISA.

Non-targeted or random antibody variant libraries can be generated forexample by error-prone PCR (Cadwell R C and Joyce G F (1994) MutagenicPCR. PCR Methods Appl. 3: S136-5140) and provide a very simple, butsometimes limited approach. Another strategy is the CDR directeddiversification of an antibody candidate. One or more positions in oneor more CDRs can be targeted specifically using for example degeneratedoligos (Thompson J et al. (1996) J. Mol. Biol. 256: 77-88) trinucleotidemutagenesis (TRIM) (Kayushin A L et al. (1996) Nucleic Acids Res. 24:3748-3755) or any other approach known to the art.

Accordingly, in another embodiment, the invention provides isolatedβ-klotho-binding antibodies, or antigen-binding fragments thereof,consisting of a heavy chain variable region having a VH CDR1 regionconsisting of an amino acid sequence selected from the group having SEQID NOs: 3, 23, 43, and 63 or an amino acid sequence having one, two,three, four or five amino acid substitutions, deletions or additions ascompared to SEQ ID NOs: 3, 23, 43, and 63; a VH CDR2 region having anamino acid sequence selected from the group consisting of SEQ ID NOs: 4,24, 44, and 64 or an amino acid sequence having one, two, three, four orfive amino acid substitutions, deletions or additions as compared to SEQID NOs: 4, 24, 44, and 64; a VH CDR3 region having an amino acidsequence selected from the group consisting of SEQ ID NOs: 5, 25, 45,and 65, or an amino acid sequence having one, two, three, four or fiveamino acid substitutions, deletions or additions as compared to SEQ IDNOs: 5, 25, 45, and 65; a VL CDR1 region having an amino acid sequenceselected from the group consisting of SEQ ID NOs: 13, 33, 53, and 73, oran amino acid sequence having one, two, three, four or five amino acidsubstitutions, deletions or additions as compared to SEQ ID NOs: 13, 33,53, and 73; a VL CDR2 region having an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 14, 34, 54, and 74, or an amino acidsequence having one, two, three, four or five amino acid substitutions,deletions or additions as compared to SEQ ID NOs: 14, 34, 54, and 74;and a VL CDR3 region having an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 15, 35, 55, and 75, or an amino acidsequence having one, two, three, four or five amino acid substitutions,deletions or additions as compared to SEQ ID NOs: 15, 35, 55, and 75.

Accordingly, in another embodiment, the invention provides isolatedβ-klotho-binding antibodies, or antigen-binding fragments thereof,consisting of a heavy chain variable region having a VH CDR1 regionconsisting of an amino acid sequence selected from the group having SEQID NOs: 6, 26, 46, and 66 or an amino acid sequence having one, two,three, four or five amino acid substitutions, deletions or additions ascompared to SEQ ID NOs: 6, 26, 46, and 66; a VH CDR2 region having anamino acid sequence selected from the group consisting of SEQ ID NOs: 7,27, 47, and 67 or an amino acid sequence having one, two, three, four orfive amino acid substitutions, deletions or additions as compared to SEQID NOs: 7, 27, 47, and 67; a VH CDR3 region having an amino acidsequence selected from the group consisting of SEQ ID NOs: 8, 28, 48,and 68, or an amino acid sequence having one, two, three, four or fiveamino acid substitutions, deletions or additions as compared to SEQ IDNOs: 8, 28, 48, and 68; a VL CDR1 region having an amino acid sequenceselected from the group consisting of SEQ ID NOs: 13, 33, 53, and 73, oran amino acid sequence having one, two, three, four or five amino acidsubstitutions, deletions or additions as compared to SEQ ID NOs: 13, 33,53, and 73; a VL CDR2 region having an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 14, 34, 54, and 74, or an amino acidsequence having one, two, three, four or five amino acid substitutions,deletions or additions as compared to SEQ ID NOs: 14, 34, 54, and 74;and a VL CDR3 region having an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 15, 35, 55, and 75, or an amino acidsequence having one, two, three, four or five amino acid substitutions,deletions or additions as compared to SEQ ID NOs: 15, 35, 55, and 75.

Grafting Antigen-Binding Domains into Alternative Frameworks orScaffolds

A wide variety of antibody/immunoglobulin frameworks or scaffolds can beemployed so long as the resulting polypeptide includes at least onebinding region which specifically binds to β-klotho. Such frameworks orscaffolds include the 5 main idiotypes of human immunoglobulins, orfragments thereof, and include immunoglobulins of other animal species,preferably having humanized aspects. Single heavy-chain antibodies suchas those identified in camelids are of particular interest in thisregard. Novel frameworks, scaffolds and fragments continue to bediscovered and developed by those skilled in the art.

In one aspect, the invention pertains to generating non-immunoglobulinbased antibodies using non-immunoglobulin scaffolds onto which CDRs ofthe invention can be grafted. Known or future non-immunoglobulinframeworks and scaffolds may be employed, as long as they comprise abinding region specific for the target β-klotho protein. Knownnon-immunoglobulin frameworks or scaffolds include, but are not limitedto, fibronectin (Compound Therapeutics, Inc., Waltham, Mass.), ankyrin(Molecular Partners AG, Zurich, Switzerland), domain antibodies(Domantis, Ltd., Cambridge, Mass., and Ablynx nv, Zwijnaarde, Belgium),lipocalin (Pieris Proteolab AG, Freising, Germany), small modularimmuno-pharmaceuticals (Trubion Pharmaceuticals Inc., Seattle, Wash.),maxybodies (Avidia, Inc., Mountain View, Calif.), Protein A (AffibodyAG, Sweden), and affilin (gamma-crystallin or ubiquitin) (Scil ProteinsGmbH, Halle, Germany).

The fibronectin scaffolds are based on fibronectin type III domain(e.g., the tenth module of the fibronectin type III (10 Fn3 domain)).The fibronectin type III domain has 7 or 8 beta strands which aredistributed between two beta sheets, which themselves pack against eachother to form the core of the protein, and further containing loops(analogous to CDRs) which connect the beta strands to each other and aresolvent exposed. There are at least three such loops at each edge of thebeta sheet sandwich, where the edge is the boundary of the proteinperpendicular to the direction of the beta strands (see U.S. Pat. No.6,818,418). These fibronectin-based scaffolds are not an immunoglobulin,although the overall fold is closely related to that of the smallestfunctional antibody fragment, the variable region of the heavy chain,which comprises the entire antigen recognition unit in camel and llamaIgG. Because of this structure, the non-immunoglobulin antibody mimicsantigen-binding properties that are similar in nature and affinity tothose of antibodies. These scaffolds can be used in a loop randomizationand shuffling strategy in vitro that is similar to the process ofaffinity maturation of antibodies in vivo. These fibronectin-basedmolecules can be used as scaffolds where the loop regions of themolecule can be replaced with CDRs of the invention using standardcloning techniques.

The ankyrin technology is based on using proteins with ankyrin derivedrepeat modules as scaffolds for bearing variable regions which can beused for binding to different targets. The ankyrin repeat module is a 33amino acid polypeptide consisting of two anti-parallel α-helices and aβ-turn. Binding of the variable regions is mostly optimized by usingribosome display.

Avimers are derived from natural A-domain containing protein such asLRP-1. These domains are used by nature for protein-protein interactionsand in human over 250 proteins are structurally based on A-domains.Avimers consist of a number of different “A-domain” monomers (2-10)linked via amino acid linkers. Avimers can be created that can bind tothe target antigen using the methodology described in, for example, U.S.Patent Application Publication Nos. 20040175756; 20050053973;20050048512; and 20060008844.

Affibody affinity ligands are small, simple proteins composed of athree-helix bundle based on the scaffold of one of the IgG-bindingdomains of Protein A. Protein A is a surface protein from the bacteriumStaphylococcus aureus. This scaffold domain consists of 58 amino acids,13 of which are randomized to generate affibody libraries with a largenumber of ligand variants (See e.g., U.S. Pat. No. 5,831,012). Affibodymolecules mimic antibodies, they have a molecular weight of 6 kDa,compared to the molecular weight of antibodies, which is 150 kDa. Inspite of its small size, the binding site of affibody molecules issimilar to that of an antibody.

Anticalins are products developed by the company Pieris ProteoLab AG.They are derived from lipocalins, a widespread group of small and robustproteins that are usually involved in the physiological transport orstorage of chemically sensitive or insoluble compounds. Several naturallipocalins occur in human tissues or body liquids. The proteinarchitecture is reminiscent of immunoglobulins, with hypervariable loopson top of a rigid framework. However, in contrast with antibodies ortheir recombinant fragments, lipocalins are composed of a singlepolypeptide chain with 160 to 180 amino acid residues, being justmarginally bigger than a single immunoglobulin domain. The set of fourloops, which makes up the binding pocket, shows pronounced structuralplasticity and tolerates a variety of side chains. The binding site canthus be reshaped in a proprietary process in order to recognizeprescribed target molecules of different shape with high affinity andspecificity. One protein of lipocalin family, the bilin-binding protein(BBP) of Pieris Brassicae has been used to develop anticalins bymutagenizing the set of four loops. One example of a patent applicationdescribing anticalins is in PCT Publication No. WO 199916873.

Affilin molecules are small non-immunoglobulin proteins which aredesigned for specific affinities towards proteins and small molecules.New affilin molecules can be very quickly selected from two libraries,each of which is based on a different human derived scaffold protein.Affilin molecules do not show any structural homology to immunoglobulinproteins. Currently, two affilin scaffolds are employed, one of which isgamma crystalline, a human structural eye lens protein and the other is“ubiquitin” superfamily proteins. Both human scaffolds are very small,show high temperature stability and are almost resistant to pH changesand denaturing agents. This high stability is mainly due to the expandedbeta sheet structure of the proteins. Examples of gamma crystallinederived proteins are described in WO200104144 and examples of“ubiquitin-like” proteins are described in WO2004106368.

Protein epitope mimetics (PEM) are medium-sized, cyclic, peptide-likemolecules (MW 1-2 kDa) mimicking beta-hairpin secondary structures ofproteins, the major secondary structure involved in protein-proteininteractions.

The present invention provides fully human antibodies that specificallybind to a β-klotho protein. Compared to the chimeric or humanizedantibodies, the human β-klotho-binding antibodies of the invention havefurther reduced antigenicity when administered to human subjects.

Camelid Antibodies

Antibody proteins obtained from members of the camel and dromedary(Camelus bactrianus and Calelus dromaderius) family including new worldmembers such as llama species (Lama paccos, Lama glama and Lama vicugna)have been characterized with respect to size, structural complexity andantigenicity for human subjects. Certain IgG antibodies from this familyof mammals as found in nature lack light chains, and are thusstructurally distinct from the typical four chain quaternary structurehaving two heavy and two light chains, for antibodies from otheranimals. See PCT/EP93/02214 (WO 94/04678 published 3 Mar. 1994).

A region of the camelid antibody which is the small single variabledomain identified as VHH can be obtained by genetic engineering to yielda small protein having high affinity for a target, resulting in a lowmolecular weight antibody-derived protein known as a “camelid nanobody”.See U.S. Pat. No. 5,759,808 issued Jun. 2, 1998; see also Stijlemans, B.et al., 2004 J Biol Chem 279: 1256-1261; Dumoulin, M. et al., 2003Nature 424: 783-788; Pleschberger, M. et al. 2003 Bioconjugate Chem 14:440-448; Cortez-Retamozo, V. et al. 2002 Int J Cancer 89: 456-62; andLauwereys, M. et al. 1998 EMBO J 17: 3512-3520. Engineered libraries ofcamelid antibodies and antibody fragments are commercially available,for example, from Ablynx, Ghent, Belgium. As with other antibodies ofnon-human origin, an amino acid sequence of a camelid antibody can bealtered recombinantly to obtain a sequence that more closely resembles ahuman sequence, i.e., the nanobody can be “humanized”. Thus the naturallow antigenicity of camelid antibodies to humans can be further reduced.

The camelid nanobody has a molecular weight approximately one-tenth thatof a human IgG molecule, and the protein has a physical diameter of onlya few nanometers. One consequence of the small size is the ability ofcamelid nanobodies to bind to antigenic sites that are functionallyinvisible to larger antibody proteins, i.e., camelid nanobodies areuseful as reagents detect antigens that are otherwise cryptic usingclassical immunological techniques, and as possible therapeutic agents.Thus yet another consequence of small size is that a camelid nanobodycan inhibit as a result of binding to a specific site in a groove ornarrow cleft of a target protein, and hence can serve in a capacity thatmore closely resembles the function of a classical low molecular weightdrug than that of a classical antibody.

The low molecular weight and compact size further result in camelidnanobodies being extremely thermostable, stable to extreme pH and toproteolytic digestion, and poorly antigenic. Another consequence is thatcamelid nanobodies readily move from the circulatory system intotissues, and even cross the blood-brain barrier and can treat disordersthat affect nervous tissue. Nanobodies can further facilitated drugtransport across the blood brain barrier. See U.S. patent application20040161738 published Aug. 19, 2004. These features combined with thelow antigenicity to humans indicate great therapeutic potential.Further, these molecules can be fully expressed in prokaryotic cellssuch as E. coli and are expressed as fusion proteins with bacteriophageand are functional.

Accordingly, a feature of the present invention is a camelid antibody ornanobody having high affinity for β-klotho. In certain embodimentsherein, the camelid antibody or nanobody is naturally produced in thecamelid animal, i.e., is produced by the camelid following immunizationwith β-klotho or a peptide fragment thereof, using techniques describedherein for other antibodies. Alternatively, the β-klotho-binding camelidnanobody is engineered, i.e., produced by selection for example from alibrary of phage displaying appropriately mutagenized camelid nanobodyproteins using panning procedures with β-klotho as a target as describedin the examples herein. Engineered nanobodies can further be customizedby genetic engineering to have a half life in a recipient subject offrom 45 minutes to two weeks. In a specific embodiment, the camelidantibody or nanobody is obtained by grafting the CDRs sequences of theheavy or light chain of the human antibodies of the invention intonanobody or single domain antibody framework sequences, as described forexample in PCT/EP93/02214.

Bispecific Molecules and Multivalent Antibodies

In another aspect, the present invention features bispecific ormultispecific molecules comprising a β-klotho-binding antibody, or afragment thereof, of the invention. An antibody of the invention, orantigen-binding regions thereof, can be derivatized or linked to anotherfunctional molecule, e.g., another peptide or protein (e.g., anotherantibody or ligand for a receptor) to generate a bispecific moleculethat binds to at least two different binding sites or target molecules.The antibody of the invention may in fact be derivatized or linked tomore than one other functional molecule to generate multi-specificmolecules that bind to more than two different binding sites and/ortarget molecules; such multi-specific molecules are also intended to beencompassed by the term “bispecific molecule” as used herein. To createa bispecific molecule of the invention, an antibody of the invention canbe functionally linked (e.g., by chemical coupling, genetic fusion,noncovalent association or otherwise) to one or more other bindingmolecules, such as another antibody, antibody fragment, peptide orbinding mimetic, such that a bispecific molecule results.

Accordingly, the present invention includes bispecific moleculescomprising at least one first binding specificity for β-klotho and asecond binding specificity for a second target epitope. For example, thesecond target epitope is another epitope of β-klotho different from thefirst target epitope.

Additionally, for the invention in which the bispecific molecule ismulti-specific, the molecule can further include a third bindingspecificity, in addition to the first and second target epitope.

In one embodiment, the bispecific molecules of the invention comprise asa binding specificity at least one antibody, or an antibody fragmentthereof, including, e.g., a Fab, Fab′, F(ab′)2, Fv, or a single chainFv. The antibody may also be a light chain or heavy chain dimer, or anyminimal fragment thereof such as a Fv or a single chain construct asdescribed in Ladner et al. U.S. Pat. No. 4,946,778.

Diabodies are bivalent, bispecific molecules in which VH and VL domainsare expressed on a single polypeptide chain, connected by a linker thatis too short to allow for pairing between the two domains on the samechain. The VH and VL domains pair with complementary domains of anotherchain, thereby creating two antigen-binding sites (see e.g., Holliger etal., 1993 Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak et al., 1994Structure 2:1121-1123). Diabodies can be produced by expressing twopolypeptide chains with either the structure VHA-VLB and VHB-VLA (VH-VLconfiguration), or VLA-VHB and VLB-VHA (VL-VH configuration) within thesame cell. Most of them can be expressed in soluble form in bacteria.Single chain diabodies (scDb) are produced by connecting the twodiabody-forming polypeptide chains with linker of approximately 15 aminoacid residues (see Holliger and Winter, 1997 Cancer Immunol.Immunother., 45(3-4):128-30; Wu et al., 1996 Immunotechnology,2(1):21-36). scDb can be expressed in bacteria in soluble, activemonomeric form (see Holliger and Winter, 1997 Cancer Immunol.Immunother., 45(34): 128-30; Wu et al., 1996 Immunotechnology,2(1):21-36; Pluckthun and Pack, 1997 Immunotechnology, 3(2): 83-105;Ridgway et al., 1996 Protein Eng., 9(7):617-21). A diabody can be fusedto Fc to generate a “di-diabody” (see Lu et al., 2004 J. Biol. Chem.,279(4):2856-65).

Other antibodies which can be employed in the bispecific molecules ofthe invention are murine, chimeric and humanized monoclonal antibodies.

Bispecific molecules can be prepared by conjugating the constituentbinding specificities, using methods known in the art. For example, eachbinding specificity of the bispecific molecule can be generatedseparately and then conjugated to one another. When the bindingspecificities are proteins or peptides, a variety of coupling orcross-linking agents can be used for covalent conjugation. Examples ofcross-linking agents include protein A, carbodiimide,N-succinimidyl-S-acetyl-thioacetate (SATA),5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide(oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), andsulfosuccinimidyl 4-(N-maleimidomethyl) cyclohaxane-1-carboxylate(sulfo-SMCC) (see e.g., Karpovsky et al., 1984 J. Exp. Med. 160:1686;Liu, M A et al., 1985 Proc. Natl. Acad. Sci. USA 82:8648). Other methodsinclude those described in Paulus, 1985 Behring Ins. Mitt. No. 78,118-132; Brennan et al., 1985 Science 229:81-83), and Glennie et al.,1987 J. Immunol. 139: 2367-2375). Conjugating agents are SATA andsulfo-SMCC, both available from Pierce Chemical Co. (Rockford, Ill.).

When the binding specificities are antibodies, they can be conjugated bysulfhydryl bonding of the C-terminus hinge regions of the two heavychains. In a particularly embodiment, the hinge region is modified tocontain an odd number of sulfhydryl residues, for example one, prior toconjugation.

Alternatively, both binding specificities can be encoded in the samevector and expressed and assembled in the same host cell. This method isparticularly useful where the bispecific molecule is a mAb×mAb, mAb×Fab,Fab×F(ab′)2 or ligand x Fab fusion protein. A bispecific molecule of theinvention can be a single chain molecule comprising one single chainantibody and a binding determinant, or a single chain bispecificmolecule comprising two binding determinants. Bispecific molecules maycomprise at least two single chain molecules. Methods for preparingbispecific molecules are described for example in U.S. Pat. Nos.5,260,203; 5,455,030; 4,881,175; 5,132,405; 5,091,513; 5,476,786;5,013,653; 5,258,498; and 5,482,858.

Binding of the bispecific molecules to their specific targets can beconfirmed by, for example, enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (REA), FACS analysis, bioassay (e.g., growthinhibition), or Western Blot assay. Each of these assays generallydetects the presence of protein-antibody complexes of particularinterest by employing a labeled reagent (e.g., an antibody) specific forthe complex of interest.

In another aspect, the present invention provides multivalent compoundscomprising at least two identical or different antigen-binding portionsof the antibodies of the invention binding to β-klotho. Theantigen-binding portions can be linked together via protein fusion orcovalent or non covalent linkage. Alternatively, methods of linkage havebeen described for the bispecfic molecules. Tetravalent compounds can beobtained for example by cross-linking antibodies of the antibodies ofthe invention with an antibody that binds to the constant regions of theantibodies of the invention, for example the Fe or hinge region.

Trimerizing domain are described for example in Borean patent EP 1 012280B1. Pentamerizing modules are described for example inPCT/EP97/05897.

Antibodies with Extended Half Life

The present invention provides for antibodies that specifically bind tobeta-klotho protein which have an extended half-life in vivo.

Many factors may affect a protein's half life in vivo. For examples,kidney filtration, metabolism in the liver, degradation by proteolyticenzymes (proteases), and immunogenic responses (e.g., proteinneutralization by antibodies and uptake by macrophages and dendriticcells). A variety of strategies can be used to extend the half life ofthe antibodies of the present invention. For example, by chemicallinkage to polyethyleneglycol (PEG), reCODE PEG, antibody scaffold,polysialic acid (PSA), hydroxyethyl starch (HES), albumin-bindingligands, and carbohydrate shields; by genetic fusion to proteins bindingto serum proteins, such as albumin, IgG, FcRn, and transferring; bycoupling (genetically or chemically) to other binding moieties that bindto serum proteins, such as nanobodies, Fabs, DARPins, avimers,affibodies, and anticalins; by genetic fusion to rPEG, albumin, domainof albumin, albumin-binding proteins, and Fc; or by incorporation intonanocarriers, slow release formulations, or medical devices.

To prolong the serum circulation of antibodies in vivo, inert polymermolecules such as high molecular weight PEG can be attached to theantibodies or a fragment thereof with or without a multifunctionallinker either through site-specific conjugation of the PEG to the N- orC-terminus of the antibodies or via epsilon-amino groups present onlysine residues. To pegylate an antibody, the antibody, or fragmentthereof, typically is reacted with polyethylene glycol (PEG), such as areactive ester or aldehyde derivative of PEG, under conditions in whichone or more PEG groups become attached to the antibody or antibodyfragment. The pegylation can be carried out by an acylation reaction oran alkylation reaction with a reactive PEG molecule (or an analogousreactive water-soluble polymer). As used herein, the term “polyethyleneglycol” is intended to encompass any of the forms of PEG that have beenused to derivatize other proteins, such as mono (C1-C10) alkoxy- oraryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certainembodiments, the antibody to be pegylated is an aglycosylated antibody.Linear or branched polymer derivatization that results in minimal lossof biological activity will be used. The degree of conjugation can beclosely monitored by SDS-PAGE and mass spectrometry to ensure properconjugation of PEG molecules to the antibodies. Unreacted PEG can beseparated from antibody-PEG conjugates by size-exclusion or byion-exchange chromatography. PEG-derivatized antibodies can be testedfor binding activity as well as for in vivo efficacy using methodswell-known to those of skill in the art, for example, by immunoassaysdescribed herein. Methods for pegylating proteins are known in the artand can be applied to the antibodies of the invention. See for example,EP 0 154 316 by Nishimura et al. and EP 0 401 384 by Ishikawa et al.

Other modified pegylation technologies include reconstituting chemicallyorthogonal directed engineering technology (ReCODE PEG), whichincorporates chemically specified side chains into biosynthetic proteinsvia a reconstituted system that includes tRNA synthetase and tRNA. Thistechnology enables incorporation of more than 30 new amino acids intobiosynthetic proteins in E. coli, yeast, and mammalian cells. The tRNAincorporates a nonnative amino acid any place an amber codon ispositioned, converting the amber from a stop codon to one that signalsincorporation of the chemically specified amino acid.

Recombinant pegylation technology (rPEG) can also be used for serumhalflife extension. This technology involves genetically fusing a300-600 amino acid unstructured protein tail to an existingpharmaceutical protein. Because the apparent molecular weight of such anunstructured protein chain is about 15-fold larger than its actualmolecular weight, the serum halflife of the protein is greatlyincreased. In contrast to traditional PEGylation, which requireschemical conjugation and repurification, the manufacturing process isgreatly simplified and the product is homogeneous.

Polysialytion is another technology, which uses the natural polymerpolysialic acid (PSA) to prolong the active life and improve thestability of therapeutic peptides and proteins. PSA is a polymer ofsialic acid (a sugar). When used for protein and therapeutic peptidedrug delivery, polysialic acid provides a protective microenvironment onconjugation. This increases the active life of the therapeutic proteinin the circulation and prevents it from being recognized by the immunesystem. The PSA polymer is naturally found in the human body. It wasadopted by certain bacteria which evolved over millions of years to coattheir walls with it. These naturally polysialylated bacteria were thenable, by virtue of molecular mimicry, to foil the body's defense system.PSA, nature's ultimate stealth technology, can be easily produced fromsuch bacteria in large quantities and with predetermined physicalcharacteristics. Bacterial PSA is completely non-immunogenic, even whencoupled to proteins, as it is chemically identical to PSA in the humanbody.

Another technology includes the use of hydroxyethyl starch (“HES”)derivatives linked to antibodies. HES is a modified natural polymerderived from waxy maize starch and can be metabolized by the body'senzymes. HES solutions are usually administered to substitute deficientblood volume and to improve the rheological properties of the blood.Hesylation of an antibody enables the prolongation of the circulationhalf-life by increasing the stability of the molecule, as well as byreducing renal clearance, resulting in an increased biological activity.By varying different parameters, such as the molecular weight of HES, awide range of HES antibody conjugates can be customized.

Antibodies having an increased half-life in vivo can also be generatedintroducing one or more amino acid modifications (i.e., substitutions,insertions or deletions) into an IgG constant domain, or FcRn bindingfragment thereof (preferably a Fc or hinge Fc domain fragment). See,e.g., International Publication No. WO 98/23289; InternationalPublication No. WO 97/34631; and U.S. Pat. No. 6,277,375.

Further, antibodies can be conjugated to albumin (e.g., human serumalbumin; HSA) in order to make the antibody or antibody fragment morestable in vivo or have a longer half life in vivo. The techniques arewell-known in the art, see, e.g., International Publication Nos. WO93/15199, WO 93/15200, and WO 01/77137; and European Patent No. EP413,622. In addition, in the context of a bispecific antibody asdescribed above, the specificities of the antibody can be designed suchthat one binding domain of the antibody binds to FGF21 while a secondbinding domain of the antibody binds to serum albumin, preferably HSA.

The strategies for increasing half life is especially useful innanobodies, fibronectin-based binders, and other antibodies or proteinsfor which increased in vivo half life is desired.

Antibody Conjugates

The present invention provides antibodies or fragments thereof thatspecifically bind to a β-klotho protein recombinantly fused orchemically conjugated (including both covalent and non-covalentconjugations) to a heterologous protein or polypeptide (or fragmentthereof, preferably to a polypeptide of at least 10, at least 20, atleast 30, at least 40, at least 50, at least 60, at least 70, at least80, at least 90 or at least 100 amino acids) to generate fusionproteins. In particular, the invention provides fusion proteinscomprising an antigen-binding fragment of an antibody described herein(e.g., a Fab fragment, Fd fragment, Fv fragment, F(ab)2 fragment, a VHdomain, a VH CDR, a VL domain or a VL CDR) and a heterologous protein,polypeptide, or peptide. Methods for fusing or conjugating proteins,polypeptides, or peptides to an antibody or an antibody fragment areknown in the art. See, e.g., U.S. Pat. Nos. 5,336,603, 5,622,929,5,359,046, 5,349,053, 5,447,851, and 5,112,946; European Patent Nos. EP307,434 and EP 367,166; International Publication Nos. WO 96/04388 andWO 91/06570; Ashkenazi et al., 1991, Proc. Natl. Acad. Sci. USA 88:10535-10539; Zheng et al., 1995, J. Immunol. 154:5590-5600; and Vil etal., 1992, Proc. Natl. Acad. Sci. USA 89:11337-11341.

Additional fusion proteins may be generated through the techniques ofgene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling(collectively referred to as “DNA shuffling”). DNA shuffling may beemployed to alter the activities of antibodies of the invention orfragments thereof (e.g., antibodies or fragments thereof with higheraffinities and lower dissociation rates). See, generally, U.S. Pat. Nos.5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458; Patten etal., 1997, Curr. Opinion Biotechnol. 8:724-33; Harayama, 1998, TrendsBiotechnol. 16(2):76-82; Hansson, et al., 1999, J. Mol. Biol.287:265-76; and Lorenzo and Blasco, 1998, Biotechniques 24(2):308-313(each of these patents and publications are hereby incorporated byreference in its entirety). Antibodies or fragments thereof, or theencoded antibodies or fragments thereof, may be altered by beingsubjected to random mutagenesis by error-prone PCR, random nucleotideinsertion or other methods prior to recombination. A polynucleotideencoding an antibody or fragment thereof that specifically binds to aβ-klotho protein may be recombined with one or more components, motifs,sections, parts, domains, fragments, etc. of one or more heterologousmolecules.

Moreover, the antibodies or fragments thereof can be fused to markersequences, such as a peptide to facilitate purification. In preferredembodiments, the marker amino acid sequence is a hexa-histidine peptide(SEQ ID NO: 274), such as the tag provided in a pQE vector (QIAGEN,Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, manyof which are commercially available. As described in Gentz et al., 1989,Proc. Natl. Acad. Sci. USA 86:821-824, for instance, hexa-histidine (SEQID NO: 274) provides for convenient purification of the fusion protein.Other peptide tags useful for purification include, but are not limitedto, the hemagglutinin (“HA”) tag, which corresponds to an epitopederived from the influenza hemagglutinin protein (Wilson et al., 1984,Cell 37:767), and the “flag” tag.

In other embodiments, antibodies of the present invention or fragmentsthereof conjugated to a diagnostic or detectable agent. Such antibodiescan be useful for monitoring or prognosing the onset, development,progression and/or severity of a disease or disorder as part of aclinical testing procedure, such as determining the efficacy of aparticular therapy. Such diagnosis and detection can accomplished bycoupling the antibody to detectable substances including, but notlimited to, various enzymes, such as, but not limited to, horseradishperoxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; prosthetic groups, such as, but not limited to,streptavidin/biotin and avidin/biotin; fluorescent materials, such as,but not limited to, umbelliferone, fluorescein, fluoresceinisothiocynate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; luminescent materials, such as, but notlimited to, luminol; bioluminescent materials, such as but not limitedto, luciferase, luciferin, and aequorin; radioactive materials, such as,but not limited to, iodine (131I, 125I, 123I, and 121I,), carbon (14C),sulfur (35S), tritium (3H), indium (115In, 113In, 112In, and 111In,),technetium (99Tc), thallium (201Ti), gallium (68Ga, 67Ga), palladium(103Pd), molybdenum (99Mo), xenon (133Xe), fluorine (18F), 153Sm, 177Lu,159Gd, 149Pm, 140La, 175Yb, 166Ho, 90Y, 47Sc, 186Re, 188Re, 142Pr,105Rh, 97Ru, 68Ge, 57Co, 65Zn, 85Sr, 32P, 153Gd, 169Yb, 51Cr, 54Mn,75Se, 113Sn, and 117Tin; and positron emitting metals using variouspositron emission tomographies, and noradioactive paramagnetic metalions.

The present invention further encompasses uses of antibodies orfragments thereof conjugated to a therapeutic moiety. An antibody orfragment thereof may be conjugated to a therapeutic moiety such as acytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent ora radioactive metal ion, e.g., alpha-emitters. A cytotoxin or cytotoxicagent includes any agent that is detrimental to cells.

Further, an antibody or fragment thereof may be conjugated to atherapeutic moiety or drug moiety that modifies a given biologicalresponse. Therapeutic moieties or drug moieties are not to be construedas limited to classical chemical therapeutic agents. For example, thedrug moiety may be a protein, peptide, or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, cholera toxin, ordiphtheria toxin; a protein such as tumor necrosis factor, α-interferon,β-interferon, nerve growth factor, platelet derived growth factor,tissue plasminogen activator, an apoptotic agent, an anti-angiogenicagent; or, a biological response modifier such as, for example, alymphokine.

Moreover, an antibody can be conjugated to therapeutic moieties such asa radioactive metal ion, such as alpha-emitters such as 213Bi ormacrocyclic chelators useful for conjugating radiometal ions, includingbut not limited to, 131In, 131LU, 131Y, 131Ho, 131Sm, to polypeptides.In certain embodiments, the macrocyclic chelator is1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA) whichcan be attached to the antibody via a linker molecule. Such linkermolecules are commonly known in the art and described in Denardo et al.,1998, Clin Cancer Res. 4(10):2483-90; Peterson et al., 1999, Bioconjug.Chem. 10(4):553-7; and Zimmerman et al., 1999, Nucl. Med. Biol.26(8):943-50, each incorporated by reference in their entireties.

Techniques for conjugating therapeutic moieties to antibodies are wellknown, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies 84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., 1982,Immunol. Rev. 62:119-58.

Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

Methods of Producing Antibodies of the Invention

Nucleic Acids Encoding the Antibodies

The invention provides substantially purified nucleic acid moleculeswhich encode polypeptides comprising segments or domains of theβ-klotho-binding antibody chains described above. Some of the nucleicacids of the invention comprise the nucleotide sequence encoding theheavy chain variable region shown in SEQ ID NO: 10, 30, 50, or 70,and/or the nucleotide sequence encoding the light chain variable regionshown in SEQ ID NO: 20, 40, 60, or 80. In a specific embodiment, thenucleic acid molecules are those identified in Table 1. Some othernucleic acid molecules of the invention comprise nucleotide sequencesthat are substantially identical (e.g., at least 65, 80%, 95%, or 99%)to the nucleotide sequences of those identified in Table 1. Whenexpressed from appropriate expression vectors, polypeptides encoded bythese polynucleotides are capable of exhibiting FGF21 antigen-bindingcapacity.

Also provided in the invention are polynucleotides which encode at leastone CDR region and usually all three CDR regions from the heavy or lightchain of the β-klotho-binding antibody set forth above. Some otherpolynucleotides encode all or substantially all of the variable regionsequence of the heavy chain and/or the light chain of theβ-klotho-binding antibody set forth above. Because of the degeneracy ofthe code, a variety of nucleic acid sequences will encode each of theimmunoglobulin amino acid sequences.

The nucleic acid molecules of the invention can encode both a variableregion and a constant region of the antibody. Some of nucleic acidsequences of the invention comprise nucleotides encoding a heavy chainsequence that is substantially identical (e.g., at least 80%, 90%, or99%) to the heavy chain sequence set forth in SEQ ID NO: 11, 31, 51, or71. Some other nucleic acid sequences comprising nucleotide encoding alight chain sequence that is substantially identical (e.g., at least80%, 90%, or 99%) to the light chain sequence set forth in SEQ ID NO:21, 41, 61, or 81.

The polynucleotide sequences can be produced by de novo solid-phase DNAsynthesis or by PCR mutagenesis of an existing sequence (e.g., sequencesas described in the Examples below) encoding a β-klotho-binding antibodyor its binding fragment. Direct chemical synthesis of nucleic acids canbe accomplished by methods known in the art, such as the phosphotriestermethod of Narang et al., 1979, Meth. Enzymol. 68:90; the phosphodiestermethod of Brown et al., Meth. Enzymol. 68:109, 1979; thediethylphosphoramidite method of Beaucage et al., Tetra. Lett., 22:1859,1981; and the solid support method of U.S. Pat. No. 4,458,066.Introducing mutations to a polynucleotide sequence by PCR can beperformed as described in, e.g., PCR Technology: Principles andApplications for DNA Amplification, H. A. Erlich (Ed.), Freeman Press,NY, N.Y., 1992; PCR Protocols: A Guide to Methods and Applications,Innis et al. (Ed.), Academic Press, San Diego, Calif., 1990; Mattila etal., Nucleic Acids Res. 19:967, 1991; and Eckert et al., PCR Methods andApplications 1:17, 1991.

Also provided in the invention are expression vectors and host cells forproducing the β-klotho-binding antibodies described above. Variousexpression vectors can be employed to express the polynucleotidesencoding the β-klotho-binding antibody chains or binding fragments. Bothviral-based and nonviral expression vectors can be used to produce theantibodies in a mammalian host cell. Nonviral vectors and systemsinclude plasmids, episomal vectors, typically with an expressioncassette for expressing a protein or RNA, and human artificialchromosomes (see, e.g., Harrington et al., Nat Genet 15:345, 1997). Forexample, nonviral vectors useful for expression of the FGF21-bindingpolynucleotides and polypeptides in mammalian (e.g., human) cellsinclude pThioHis A, B & C, pcDNA3.1/His, pEBVHis A, B & C, (Invitrogen,San Diego, Calif.), MPSV vectors, and numerous other vectors known inthe art for expressing other proteins. Useful viral vectors includevectors based on retroviruses, adenoviruses, adenoassociated viruses,herpes viruses, vectors based on SV40, papilloma virus, HBP Epstein Barrvirus, vaccinia virus vectors and Semliki Forest virus (SFV). See, Brentet al., supra; Smith, Annu. Rev. Microbiol. 49:807, 1995; and Rosenfeldet al., Cell 68:143, 1992.

The choice of expression vector depends on the intended host cells inwhich the vector is to be expressed. Typically, the expression vectorscontain a promoter and other regulatory sequences (e.g., enhancers) thatare operably linked to the polynucleotides encoding a β-klotho-bindingantibody chain or fragment. In some embodiments, an inducible promoteris employed to prevent expression of inserted sequences except underinducing conditions. Inducible promoters include, e.g., arabinose, lacZ,metallothionein promoter or a heat shock promoter. Cultures oftransformed organisms can be expanded under noninducing conditionswithout biasing the population for coding sequences whose expressionproducts are better tolerated by the host cells. In addition topromoters, other regulatory elements may also be required or desired forefficient expression of a β-klotho-binding antibody chain or fragment.These elements typically include an ATG initiation codon and adjacentribosome binding site or other sequences. In addition, the efficiency ofexpression may be enhanced by the inclusion of enhancers appropriate tothe cell system in use (see, e.g., Scharf et al., Results Probl. CellDiffer. 20:125, 1994; and Bittner et al., Meth. Enzymol., 153:516,1987). For example, the SV40 enhancer or CMV enhancer may be used toincrease expression in mammalian host cells.

The expression vectors may also provide a secretion signal sequenceposition to form a fusion protein with polypeptides encoded by insertedFGF21-binding antibody sequences. More often, the insertedβ-klotho-binding antibody sequences are linked to a signal sequencesbefore inclusion in the vector. Vectors to be used to receive sequencesencoding β-klotho-binding antibody light and heavy chain variabledomains sometimes also encode constant regions or parts thereof. Suchvectors allow expression of the variable regions as fusion proteins withthe constant regions thereby leading to production of intact antibodiesor fragments thereof. Typically, such constant regions are human.

The host cells for harboring and expressing the β-klotho-bindingantibody chains can be either prokaryotic or eukaryotic. E. coli is oneprokaryotic host useful for cloning and expressing the polynucleotidesof the present invention. Other microbial hosts suitable for use includebacilli, such as Bacillus subtilis, and other enterobacteriaceae, suchas Salmonella, Serratia, and various Pseudomonas species. In theseprokaryotic hosts, one can also make expression vectors, which typicallycontain expression control sequences compatible with the host cell(e.g., an origin of replication). In addition, any number of a varietyof well-known promoters will be present, such as the lactose promotersystem, a tryptophan (trp) promoter system, a beta-lactamase promotersystem, or a promoter system from phage lambda. The promoters typicallycontrol expression, optionally with an operator sequence, and haveribosome binding site sequences and the like, for initiating andcompleting transcription and translation. Other microbes, such as yeast,can also be employed to express FGF21-binding polypeptides of theinvention. Insect cells in combination with baculovirus vectors can alsobe used.

In some preferred embodiments, mammalian host cells are used to expressand produce the β-klotho-binding polypeptides of the present invention.For example, they can be either a hybridoma cell line expressingendogenous immunoglobulin genes (e.g., the 1D6.C9 myeloma hybridomaclone as described in the Examples) or a mammalian cell line harboringan exogenous expression vector (e.g., the SP2/0 myeloma cellsexemplified below). These include any normal mortal or normal orabnormal immortal animal or human cell. For example, a number ofsuitable host cell lines capable of secreting intact immunoglobulinshave been developed including the CHO cell lines, various Cos celllines, HeLa cells, myeloma cell lines, transformed B-cells andhybridomas. The use of mammalian tissue cell culture to expresspolypeptides is discussed generally in, e.g., Winnacker, FROM GENES TOCLONES, VCH Publishers, N.Y., N.Y., 1987. Expression vectors formammalian host cells can include expression control sequences, such asan origin of replication, a promoter, and an enhancer (see, e.g., Queen,et al., Immunol. Rev. 89:49-68, 1986), and necessary processinginformation sites, such as ribosome binding sites, RNA splice sites,polyadenylation sites, and transcriptional terminator sequences. Theseexpression vectors usually contain promoters derived from mammaliangenes or from mammalian viruses. Suitable promoters may be constitutive,cell type-specific, stage-specific, and/or modulatable or regulatable.Useful promoters include, but are not limited to, the metallothioneinpromoter, the constitutive adenovirus major late promoter, thedexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP polIIIpromoter, the constitutive MPSV promoter, the tetracycline-inducible CMVpromoter (such as the human immediate-early CMV promoter), theconstitutive CMV promoter, and promoter-enhancer combinations known inthe art.

Methods for introducing expression vectors containing the polynucleotidesequences of interest vary depending on the type of cellular host. Forexample, calcium chloride transfection is commonly utilized forprokaryotic cells, whereas calcium phosphate treatment orelectroporation may be used for other cellular hosts. (See generallySambrook, et al., supra). Other methods include, e.g., electroporation,calcium phosphate treatment, liposome-mediated transformation, injectionand microinjection, ballistic methods, virosomes, immunoliposomes,polycation:nucleic acid conjugates, naked DNA, artificial virions,fusion to the herpes virus structural protein VP22 (Elliot and O'Hare,Cell 88:223, 1997), agent-enhanced uptake of DNA, and ex vivotransduction. For long-term, high-yield production of recombinantproteins, stable expression will often be desired. For example, celllines which stably express β-klotho-binding antibody chains or bindingfragments can be prepared using expression vectors of the inventionwhich contain viral origins of replication or endogenous expressionelements and a selectable marker gene. Following the introduction of thevector, cells may be allowed to grow for 1-2 days in an enriched mediabefore they are switched to selective media. The purpose of theselectable marker is to confer resistance to selection, and its presenceallows growth of cells which successfully express the introducedsequences in selective media. Resistant, stably transfected cells can beproliferated using tissue culture techniques appropriate to the celltype.

Generation of Monoclonal Antibodies

Monoclonal antibodies (mAbs) can be produced by a variety of techniques,including conventional monoclonal antibody methodology e.g., thestandard somatic cell hybridization technique of Kohler and Milstein,1975 Nature 256: 495. Many techniques for producing monoclonal antibodycan be employed e.g., viral or oncogenic transformation of Blymphocytes.

Animal systems for preparing hybridomas include the murine, rat andrabbit systems. Hybridoma production in the mouse is a well establishedprocedure. Immunization protocols and techniques for isolation ofimmunized splenocytes for fusion are known in the art. Fusion partners(e.g., murine myeloma cells) and fusion procedures are also known.

Chimeric or humanized antibodies of the present invention can beprepared based on the sequence of a murine monoclonal antibody preparedas described above. DNA encoding the heavy and light chainimmunoglobulins can be obtained from the murine hybridoma of interestand engineered to contain non-murine (e.g., human) immunoglobulinsequences using standard molecular biology techniques. For example, tocreate a chimeric antibody, the murine variable regions can be linked tohuman constant regions using methods known in the art (see e.g., U.S.Pat. No. 4,816,567 to Cabilly et al.). To create a humanized antibody,the murine CDR regions can be inserted into a human framework usingmethods known in the art. See e.g., U.S. Pat. No. 5,225,539 to Winter,and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 toQueen et al.

In a certain embodiment, the antibodies of the invention are humanmonoclonal antibodies. Such human monoclonal antibodies directed againstβ-klotho can be generated using transgenic or transchromosomic micecarrying parts of the human immune system rather than the mouse system.These transgenic and transchromosomic mice include mice referred toherein as HuMAb mice and KM mice, respectively, and are collectivelyreferred to herein as “human Ig mice.”

The HuMAb Mouse® (Medarex, Inc.) contains human immunoglobulin geneminiloci that encode un-rearranged human heavy (μ and γ) and κ lightchain immunoglobulin sequences, together with targeted mutations thatinactivate the endogenous μ and κ chain loci (see e.g., Lonberg, et al.,1994 Nature 368(6474): 856-859). Accordingly, the mice exhibit reducedexpression of mouse IgM or κ, and in response to immunization, theintroduced human heavy and light chain transgenes undergo classswitching and somatic mutation to generate high affinity human IgGκmonoclonal (Lonberg, N. et al., 1994 supra; reviewed in Lonberg, N.,1994 Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. andHuszar, D., 1995 Intern. Rev. Immunol. 13: 65-93, and Harding, F. andLonberg, N., 1995 Ann. N. Y. Acad. Sci. 764:536-546). The preparationand use of HuMAb mice, and the genomic modifications carried by suchmice, is further described in Taylor, L. et al., 1992 Nucleic AcidsResearch 20:6287-6295; Chen, J. et at., 1993 International Immunology 5:647-656; Tuaillon et al., 1993 Proc. Natl. Acad. Sci. USA 94:3720-3724;Choi et al., 1993 Nature Genetics 4:117-123; Chen, J. et al., 1993 EMBOJ. 12: 821-830; Tuaillon et al., 1994 J. Immunol. 152:2912-2920; Taylor,L. et al., 1994 International Immunology 579-591; and Fishwild, D. etal., 1996 Nature Biotechnology 14: 845-851, the contents of all of whichare hereby specifically incorporated by reference in their entirety. Seefurther, U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425;5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429;all to Lonberg and Kay; U.S. Pat. No. 5,545,807 to Surani et al.; PCTPublication Nos. WO 92103918, WO 93/12227, WO 94/25585, WO 97113852, WO98/24884 and WO 99/45962, all to Lonberg and Kay; and PCT PublicationNo. WO 01/14424 to Korman et al.

In another embodiment, human antibodies of the invention can be raisedusing a mouse that carries human immunoglobulin sequences on transgenesand transchomosomes such as a mouse that carries a human heavy chaintransgene and a human light chain transchromosome. Such mice, referredto herein as “KM mice”, are described in detail in PCT Publication WO02/43478 to Ishida et al.

Still further, alternative transgenic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseβ-klotho-binding antibodies of the invention. For example, analternative transgenic system referred to as the Xenomouse (Abgenix,Inc.) can be used. Such mice are described in, e.g., U.S. Pat. Nos.5,939,598; 6,075,181; 6,114,598; 6, 150,584 and 6,162,963 toKucherlapati et al.

Moreover, alternative transchromosomic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseβ-klotho-binding antibodies of the invention. For example, mice carryingboth a human heavy chain transchromosome and a human light chaintranchromosome, referred to as “TC mice” can be used; such mice aredescribed in Tomizuka et al., 2000 Proc. Natl. Acad. Sci. USA97:722-727. Furthermore, cows carrying human heavy and light chaintranschromosomes have been described in the art (Kuroiwa et al., 2002Nature Biotechnology 20:889-894) and can be used to raise FGF21-bindingantibodies of the invention.

Human monoclonal antibodies of the invention can also be prepared usingphage display methods for screening libraries of human immunoglobulingenes. Such phage display methods for isolating human antibodies areestablished in the art or described in the examples below. See forexample: U.S. Pat. Nos. 5,223,409; 5,403,484; and 5,571,698 to Ladner etal.; U.S. Pat. Nos. 5,427,908 and 5,580,717 to Dower et al.; U.S. Pat.Nos. 5,969,108 and 6,172,197 to McCafferty et al.; and U.S. Pat. Nos.5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and 6,593,081 toGriffiths et al.

Human monoclonal antibodies of the invention can also be prepared usingSCID mice into which human immune cells have been reconstituted suchthat a human antibody response can be generated upon immunization. Suchmice are described in, for example, U.S. Pat. Nos. 5,476,996 and5,698,767 to Wilson et al.

Framework or Fc Engineering

Engineered antibodies of the invention include those in whichmodifications have been made to framework residues within VH and/or VL,e.g. to improve the properties of the antibody. Typically such frameworkmodifications are made to decrease the immunogenicity of the antibody.For example, one approach is to “backmutate” one or more frameworkresidues to the corresponding germline sequence. More specifically, anantibody that has undergone somatic mutation may contain frameworkresidues that differ from the germline sequence from which the antibodyis derived. Such residues can be identified by comparing the antibodyframework sequences to the germline sequences from which the antibody isderived. To return the framework region sequences to their germlineconfiguration, the somatic mutations can be “backmutated” to thegermline sequence by, for example, site-directed mutagenesis. Such“backmutated” antibodies are also intended to be encompassed by theinvention.

Another type of framework modification involves mutating one or moreresidues within the framework region, or even within one or more CDRregions, to remove T cell—epitopes to thereby reduce the potentialimmunogenicity of the antibody. This approach is also referred to as“deimmunization” and is described in further detail in U.S. PatentPublication No. 20030153043 by Carr et al.

In addition or alternative to modifications made within the framework orCDR regions, antibodies of the invention may be engineered to includemodifications within the Fc region, typically to alter one or morefunctional properties of the antibody, such as serum half-life,complement fixation, Fc receptor binding, and/or antigen-dependentcellular cytotoxicity. Furthermore, an antibody of the invention may bechemically modified (e.g., one or more chemical moieties can be attachedto the antibody) or be modified to alter its glycosylation, again toalter one or more functional properties of the antibody. Each of theseembodiments is described in further detail below. The numbering ofresidues in the Fc region is that of the EU index of Kabat.

In one embodiment, the hinge region of CH1 is modified such that thenumber of cysteine residues in the hinge region is altered, e.g.,increased or decreased. This approach is described further in U.S. Pat.No. 5,677,425 by Bodmer et al. The number of cysteine residues in thehinge region of CH1 is altered to, for example, facilitate assembly ofthe light and heavy chains or to increase or decrease the stability ofthe antibody.

In another embodiment, the Fc hinge region of an antibody is mutated todecrease the biological half-life of the antibody. More specifically,one or more amino acid mutations are introduced into the CH2-CH3 domaininterface region of the Fc-hinge fragment such that the antibody hasimpaired Staphylococcyl protein A (SpA) binding relative to nativeFc-hinge domain SpA binding. This approach is described in furtherdetail in U.S. Pat. No. 6,165,745 by Ward et al.

In another embodiment, the antibody is modified to increase itsbiological half-life. Various approaches are possible. For example, oneor more of the following mutations can be introduced: T252L, T254S,T256F, as described in U.S. Pat. No. 6,277,375 to Ward. Alternatively,to increase the biological half life, the antibody can be altered withinthe CH1 or CL region to contain a salvage receptor binding epitope takenfrom two loops of a CH2 domain of an Fc region of an IgG, as describedin U.S. Pat. Nos. 5,869,046 and 6,121,022 by Presta et al.

In yet other embodiments, the Fc region is altered by replacing at leastone amino acid residue with a different amino acid residue to alter theeffector functions of the antibody. For example, one or more amino acidscan be replaced with a different amino acid residue such that theantibody has an altered affinity for an effector ligand but retains theantigen-binding ability of the parent antibody. The effector ligand towhich affinity is altered can be, for example, an Fc receptor or the C1component of complement. This approach is described in further detail inU.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.

In another embodiment, one or more amino acids selected from amino acidresidues can be replaced with a different amino acid residue such thatthe antibody has altered Clq binding and/or reduced or abolishedcomplement dependent cytotoxicity (CDC). This approach is described infurther detail in U.S. Pat. No. 6,194,551 by Idusogie et al.

In another embodiment, one or more amino acid residues are altered tothereby alter the ability of the antibody to fix complement. Thisapproach is described further in PCT Publication WO 94/29351 by Bodmeret al.

In yet another embodiment, the Fc region is modified to increase theability of the antibody to mediate antibody dependent cellularcytotoxicity (ADCC) and/or to increase the affinity of the antibody foran Fcγ receptor by modifying one or more amino acids. This approach isdescribed further in PCT Publication WO 00/42072 by Presta. Moreover,the binding sites on human IgG1 for FcγRI, FcγRII, FcγRIII and FcRn havebeen mapped and variants with improved binding have been described (seeShields, R. L. et al., 2001 J. Biol. Chen. 276:6591-6604).

In still another embodiment, the glycosylation of an antibody ismodified. For example, an aglycoslated antibody can be made (i.e., theantibody lacks glycosylation). Glycosylation can be altered to, forexample, increase the affinity of the antibody for “antigen”. Suchcarbohydrate modifications can be accomplished by, for example, alteringone or more sites of glycosylation within the antibody sequence. Forexample, one or more amino acid substitutions can be made that result inelimination of one or more variable region framework glycosylation sitesto thereby eliminate glycosylation at that site. Such aglycosylation mayincrease the affinity of the antibody for antigen. Such an approach isdescribed in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 byCo et al.

Additionally or alternatively, an antibody can be made that has analtered type of glycosylation, such as a hypofucosylated antibody havingreduced amounts of fucosyl residues or an antibody having increasedbisecting GlcNac structures. Such altered glycosylation patterns havebeen demonstrated to increase the ADCC ability of antibodies. Suchcarbohydrate modifications can be accomplished by, for example,expressing the antibody in a host cell with altered glycosylationmachinery. Cells with altered glycosylation machinery have beendescribed in the art and can be used as host cells in which to expressrecombinant antibodies of the invention to thereby produce an antibodywith altered glycosylation. For example, EP 1,176,195 by Hang et al.describes a cell line with a functionally disrupted FUT8 gene, whichencodes a fucosyl transferase, such that antibodies expressed in such acell line exhibit hypofucosylation. PCT Publication WO 03/035835 byPresta describes a variant CHO cell line, Lecl3 cells, with reducedability to attach fucose to Asn(297)-linked carbohydrates, alsoresulting in hypofucosylation of antibodies expressed in that host cell(see also Shields, R. L. et al., 2002 J. Biol. Chem. 277:26733-26740).PCT Publication WO 99/54342 by Umana et al. describes cell linesengineered to express glycoprotein-modifying glycosyl transferases(e.g., beta(1,4)-N acetylglucosaminyltransferase III (GnTIII)) such thatantibodies expressed in the engineered cell lines exhibit increasedbisecting GlcNac structures which results in increased ADCC activity ofthe antibodies (see also Umana et al., 1999 Nat. Biotech. 17:176-180).

Methods of Engineering Altered Antibodies

As discussed above, the β-klotho-binding antibodies having VH and VLsequences or full length heavy and light chain sequences shown hereincan be used to create new β-klotho-binding antibodies by modifying fulllength heavy chain and/or light chain sequences, VH and/or VL sequences,or the constant region(s) attached thereto. Thus, in another aspect ofthe invention, the structural features of a β-klotho-binding antibody ofthe invention are used to create structurally related β-klotho-bindingantibodies that retain at least one functional property of theantibodies of the invention, such as binding to human β-klotho and alsoactivating one or more functional properties of the FGF21-receptorcomplex (e.g., activating FGF21-receptor signaling).

For example, one or more CDR regions of the antibodies of the presentinvention, or mutations thereof, can be combined recombinantly withknown framework regions and/or other CDRs to create additional,recombinantly-engineered, β-klotho-binding antibodies of the invention,as discussed above. Other types of modifications include those describedin the previous section. The starting material for the engineeringmethod is one or more of the VH and/or VL sequences provided herein, orone or more CDR regions thereof. To create the engineered antibody, itis not necessary to actually prepare (i.e., express as a protein) anantibody having one or more of the VH and/or VL sequences providedherein, or one or more CDR regions thereof. Rather, the informationcontained in the sequence(s) is used as the starting material to createa “second generation” sequence(s) derived from the original sequence(s)and then the “second generation” sequence(s) is prepared and expressedas a protein.

Accordingly, in another embodiment, the invention provides a method forpreparing a β-klotho-binding antibody consisting of a heavy chainvariable region antibody sequence having a CDR1 sequence selected fromthe group consisting of SEQ ID NOs: 3, 23, 43, and 63, a CDR2 sequenceselected from the group consisting of SEQ ID NOs: 4, 24, 44, and 64,and/or a CDR3 sequence selected from the group consisting of SEQ ID NOs:5, 25, 45, and 65; and a light chain variable region antibody sequencehaving a CDR1 sequence selected from the group consisting of SEQ ID NOs:13, 33, 53, and 73, a CDR2 sequence selected from the group consistingof SEQ ID NOs: 14, 34, 54, and 74, and/or a CDR3 sequence selected fromthe group consisting of SEQ ID NOs: 15, 35, 55, and 75; altering atleast one amino acid residue within the heavy chain variable regionantibody sequence and/or the light chain variable region antibodysequence to create at least one altered antibody sequence; andexpressing the altered antibody sequence as a protein.

Accordingly, in another embodiment, the invention provides a method forpreparing a β-klotho-binding antibody consisting of a heavy chainvariable region antibody sequence having a CDR1 sequence selected fromthe group consisting of SEQ ID NOs: 6, 26, 46, and 66, a CDR2 sequenceselected from the group consisting of SEQ ID NOs: 7, 27, 47, and 67,and/or a CDR3 sequence selected from the group consisting of SEQ ID NOs:8, 28, 48, and 68; and a light chain variable region antibody sequencehaving a CDR1 sequence selected from the group consisting of SEQ ID NOs:16, 36, 56, and 76, a CDR2 sequence selected from the group consistingof SEQ ID NOs: 17, 37, 57, and 77, and/or a CDR3 sequence selected fromthe group consisting of SEQ ID NOs: 18, 38, 58, and 78; altering atleast one amino acid residue within the heavy chain variable regionantibody sequence and/or the light chain variable region antibodysequence to create at least one altered antibody sequence; andexpressing the altered antibody sequence as a protein.

Accordingly, in another embodiment, the invention provides a method forpreparing a β-klotho-binding antibody optimized for expression in amammalian cell consisting of: a full length heavy chain antibodysequence having a sequence selected from the group of SEQ ID NOs: 11,31, 51, or 71; and a full length light chain antibody sequence having asequence selected from the group of 21, 41, 61, or 81; altering at leastone amino acid residue within the full length heavy chain antibodysequence and/or the full length light chain antibody sequence to createat least one altered antibody sequence; and expressing the alteredantibody sequence as a protein. In one embodiment, the alteration of theheavy or light chain is in the framework region of the heavy or lightchain.

The altered antibody sequence can also be prepared by screening antibodylibraries having fixed CDR3 sequences or minimal essential bindingdeterminants as described in US2005/0255552 and diversity on CDR1 andCDR2 sequences. The screening can be performed according to anyscreening technology appropriate for screening antibodies from antibodylibraries, such as phage display technology.

Standard molecular biology techniques can be used to prepare and expressthe altered antibody sequence. The antibody encoded by the alteredantibody sequence(s) is one that retains one, some or all of thefunctional properties of the β-klotho-binding antibodies describedherein, which functional properties include, but are not limited to,specifically binding to human, cynomolgus, rat, and/or mouse β-klotho;and the antibody activates FGF21-mediated signaling, e.g.,FGF21-receptor-dependent signaling, in a FGFR1c_β-klotho_HEK293 pERKcell assay.

In certain embodiments of the methods of engineering antibodies of theinvention, mutations can be introduced randomly or selectively along allor part of a β-klotho-binding antibody coding sequence and the resultingmodified β-klotho-binding antibodies can be screened for bindingactivity and/or other functional properties as described herein.Mutational methods have been described in the art. For example, PCTPublication WO 02/092780 by Short describes methods for creating andscreening antibody mutations using saturation mutagenesis, syntheticligation assembly, or a combination thereof. Alternatively, PCTPublication WO 03/074679 by Lazar et al. describes methods of usingcomputational screening methods to optimize physiochemical properties ofantibodies.

In certain embodiments of the invention antibodies have been engineeredto remove sites of deamidation. Deamidation is known to cause structuraland functional changes in a peptide or protein. Deamindation can resultin decreased bioactivity, as well as alterations in pharmacokinetics andantigenicity of the protein pharmaceutical. (Anal Chem. 2005 Mar. 1;77(5):1432-9).

In certain embodiments of the invention the antibodies have beenengineered to increase pI and improve their drug-like properties. The pIof a protein is a key determinant of the overall biophysical propertiesof a molecule. Antibodies that have low pIs have been known to be lesssoluble, less stable, and prone to aggregation. Further, thepurification of antibodies with low pI is challenging and can beproblematic especially during scale-up for clinical use. Increasing thepI of the anti-β-klotho antibodies, or Fabs, of the invention improvedtheir solubility, enabling the antibodies to be formulated at higherconcentrations (>100 mg/ml). Formulation of the antibodies at highconcentrations (e.g. >100 mg/ml) offers the advantage of being able toadminister higher doses of the antibodies into eyes of patients viaintravitreal injections, which in turn may enable reduced dosingfrequency, a significant advantage for treatment of chronic diseasesincluding cardiovascular disorders. Higher pIs may also increase theFcRn-mediated recycling of the IgG version of the antibody thus enablingthe drug to persist in the body for a longer duration, requiring fewerinjections. Finally, the overall stability of the antibodies issignificantly improved due to the higher pI resulting in longershelf-life and bioactivity in vivo. Preferably, the pI is greater thanor equal to 8.2.

The functional properties of the altered antibodies can be assessedusing standard assays available in the art and/or described herein, suchas those set forth in the Examples (e.g., ELISAs).

Prophylactic and Therapeutic Uses

Antibodies that bind β-klotho as described herein, can be used at atherapeutically useful concentration for the treatment of a disease ordisorder associated with aberrant FGF21 signaling (e.g., aberrantactivation of FGF21-mediated signaling and/or FGF21 receptor signaling),by administering to a subject in need thereof an effective amount of theantibodies or antigen-binding fragments of the invention. The presentinvention provides a method of treating FGF21-associated metabolicdisorders by administering to a subject in need thereof an effectiveamount of the antibodies of the invention. The present inventionprovides a method of treating FGF21-associated cardiovascular disordersby administering to a subject in need thereof an effective amount of theantibodies of the invention.

The antibodies of the invention can be used, inter alia, to preventtreat, prevent, and improve FGF21 associated conditions or disorders,including but not limited to metabolic, endocrine, and cardiovasculardisorders, such as obesity, type 1 and type 2 diabetes mellitus,pancreatitis, dyslipidemia, nonalcoholic fatty liver disease (NAFLD),nonalcoholic steatohepatitis (NASH), insulin resistance,hyperinsulinemia, glucose intolerance, hyperglycemia, metabolicsyndrome, acute myocardial infarction, hypertension, cardiovasculardisease, atherosclerosis, peripheral arterial disease, stroke, heartfailure, coronary heart disease, kidney disease, diabetic complications,neuropathy, gastroparesis, disorders associated with severe inactivatingmutations in the insulin receptor, and other metabolic disorders, and inreducing the mortality and morbidity of critically ill patients.

The antibodies of the invention can also be used in combination withother agents for the prevention, treatment, or improvement of FGF21associated disorders. For example, statin therapies may be used incombination with the FGF21 mimetic antibodies and antigen-bindingfragments of the invention for the treatment of patients withcardiovascular or metabolic disorders.

In particular aspects, provided herein is a method of reducing bodyweight (e.g., by at least 4%, at least 5%, at least 6%, at least 7%, atleast 8%, at least 9%, at least 10%, at least 12%, at least 15%, or atleast 20%) comprising administering to a subject in need thereof aneffective amount of a pharmaceutical composition comprising an antibodyor antigen-binding fragment described herein which binds β-klotho and iscapable of increases the activity of 0-klotho and FGFR1c.

In particular aspects, provided herein is a method of reducing appetiteor food intake (e.g., by at least 4%, at least 5%, at least 6%, at least7%, at least 8%, at least 9%, at least 10%, at least 12%, at least 15%,or at least 20%) comprising administering to a subject in need thereofan effective amount of a pharmaceutical composition comprising anantibody or antigen-binding fragment described herein which bindsβ-klotho and is capable of increases the activity of β-klotho andFGFR1c.

In particular aspects, provided herein is a method of reducing (e.g., byat least 4%, at least 5%, at least 6%, at least 7%, at least 8%, atleast 9%, at least 10%, at least 12%, at least 15%, or at least 20%)plasma triglyceride (TG) concentrations or plasma total cholesterol (TC)concentrations in a subject, comprising administering to a subject inneed thereof an effective amount of a pharmaceutical compositioncomprising an antibody or antigen-binding fragment described hereinwhich binds β-klotho and is capable of increases the activity of0-klotho and FGFR1c.

In specific aspects, the subject is afflicted with a metabolic disorder,such as obesity, type 1 and type 2 diabetes mellitus, pancreatitis,dyslipidemia, nonalcoholic steatohepatitis (NASH), insulin resistance,hyperinsulinemia, glucose intolerance, hyperglycemia, and metabolicsyndrome. In specific aspects, the subject is afflicted with acardiovascular disorder.

Pharmaceutical Compositions

The invention provides pharmaceutical compositions comprising theβ-klotho-binding antibodies (intact or binding fragments) formulatedtogether with a pharmaceutically acceptable carrier. The compositionscan additionally contain one or more other therapeutic agents that aresuitable for treating or preventing, for example, cardiovasculardisorders. Pharmaceutically acceptable carriers enhance or stabilize thecomposition, or can be used to facilitate preparation of thecomposition. Pharmaceutically acceptable carriers include solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like that arephysiologically compatible.

A pharmaceutical composition of the present invention can beadministered by a variety of methods known in the art. The route and/ormode of administration vary depending upon the desired results. It ispreferred that administration be intravitreal, intravenous,intramuscular, intraperitoneal, or subcutaneous, or administeredproximal to the site of the target. The pharmaceutically acceptablecarrier should be suitable for intravitreal, intravenous, intramuscular,subcutaneous, parenteral, spinal or epidermal administration (e.g., byinjection or infusion). Depending on the route of administration, theactive compound, i.e., antibody, bispecific and multispecific molecule,may be coated in a material to protect the compound from the action ofacids and other natural conditions that may inactivate the compound.

The composition should be sterile and fluid. Proper fluidity can bemaintained, for example, by use of coating such as lecithin, bymaintenance of required particle size in the case of dispersion and byuse of surfactants. In many cases, it is preferable to include isotonicagents, for example, sugars, polyalcohols such as mannitol or sorbitol,and sodium chloride in the composition. Long-term absorption of theinjectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate or gelatin.

Pharmaceutical compositions of the invention can be prepared inaccordance with methods well known and routinely practiced in the art.See, e.g., Remington: The Science and Practice of Pharmacy, MackPublishing Co., 20th ed., 2000; and Sustained and Controlled ReleaseDrug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., NewYork, 1978. Pharmaceutical compositions are preferably manufacturedunder GMP conditions. Typically, a therapeutically effective dose orefficacious dose of the β-klotho-binding antibody is employed in thepharmaceutical compositions of the invention. The β-klotho-bindingantibodies are formulated into pharmaceutically acceptable dosage formsby conventional methods known to those of skill in the art. Dosageregimens are adjusted to provide the optimum desired response (e.g., atherapeutic response). For example, a single bolus may be administered,several divided doses may be administered over time or the dose may beproportionally reduced or increased as indicated by the exigencies ofthe therapeutic situation. It is especially advantageous to formulateparenteral compositions in dosage unit form for ease of administrationand uniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subjects tobe treated; each unit contains a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention can be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level depends upon a variety of pharmacokinetic factors includingthe activity of the particular compositions of the present inventionemployed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors.

A physician or veterinarian can start doses of the antibodies of theinvention employed in the pharmaceutical composition at levels lowerthan that required to achieve the desired therapeutic effect andgradually increase the dosage until the desired effect is achieved. Ingeneral, effective doses of the compositions of the present invention,for the treatment of a cardiovascular disorders described herein varydepending upon many different factors, including means ofadministration, target site, physiological state of the patient, whetherthe patient is human or an animal, other medications administered, andwhether treatment is prophylactic or therapeutic. Treatment dosages needto be titrated to optimize safety and efficacy. In a particularembodiment, for systemic administration with an antibody, the dosageranges from about 0.0001 to 100 mg/kg, or from 0.01 to 15 mg/kg, of thehost body weight. In a specific embodiment, for intravitrealadministration with an antibody, the dosage may range from 0.1 mg/eye to5 mg/eye. For example, 0.1 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml, 0.5mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, 1.0 mg/ml, 1.1 mg/ml,1.2 mg/ml, 1.3 mg/ml, 1.4 mg/ml, 1.5 mg/ml, 1.6 mg/ml, 1.7 mg/ml, 1.8mg/ml, 1.9 mg/ml, 2.0 mg/ml, 2.1 mg/ml, 2.2 mg/ml, 2.3 mg/ml, 2.4 mg/ml,2.5 mg/ml, 2.6 mg/ml, 2.7 mg/ml, 2.8 mg/ml, 2.9 mg/ml, 3.0 mg/ml, 3.1mg/ml, 3.2 mg/ml, 3.3 mg/ml, 3.4 mg/ml, 3.5 mg/ml, 3.6 mg/ml, 3.7 mg/ml,3.8 mg/ml, 3.9 mg/ml, 4.0 mg/ml, 4.1 mg/ml, 4.2 mg/ml, 4.3 mg/ml, 4.4mg/ml, 4.5 mg/ml, 4.6 mg/ml, 4.7 mg/ml, 4.8 mg/ml, 4.9 mg/ml, or 5.0mg/ml. An exemplary treatment regime entails systemic administrationonce per every two weeks or once a month or once every 3 to 6 months. Anexemplary treatment regime entails systemic administration once perevery two weeks or once a month or once every 3 to 6 months, or asneeded (PRN).

Antibody is usually administered on multiple occasions. Intervalsbetween single dosages can be weekly, monthly or yearly. Intervals canalso be irregular as indicated by measuring blood levels ofβ-klotho-binding antibody in the patient. In addition alternative dosingintervals can be determined by a physician and administered monthly oras necessary to be efficacious. In some methods of systemicadministration, dosage is adjusted to achieve a plasma antibodyconcentration of 1-1000 μg/ml and in some methods 25-500 μg/ml.Alternatively, antibody can be administered as a sustained releaseformulation, in which case less frequent administration is required.Dosage and frequency vary depending on the half-life of the antibody inthe patient. In general, humanized antibodies show longer half life thanthat of chimeric antibodies and nonhuman antibodies. The dosage andfrequency of administration can vary depending on whether the treatmentis prophylactic or therapeutic. In prophylactic applications, arelatively low dosage is administered at relatively infrequent intervalsover a long period of time. Some patients continue to receive treatmentfor the rest of their lives. In therapeutic applications, a relativelyhigh dosage at relatively short intervals is sometimes required untilprogression of the disease is reduced or terminated, and preferablyuntil the patient shows partial or complete amelioration of symptoms ofdisease. Thereafter, the patient can be administered a prophylacticregime.

EXAMPLES

The following examples are provided to further illustrate the inventionbut not to limit its scope. Other variants of the invention will bereadily apparent to one of ordinary skill in the art and are encompassedby the appended claims.

Example 1: Preparation of Human FGFR1c β-Klotho 300.19 Cells for Use asan Antigen

300.19 cells which stably expressed the human FGFR1c (1-386 aa) andβ-klotho were generated for use as a whole cell antigen. The full-lengthcDNA encoding human β-klotho (GenBank Accession number NM_175737) wascloned into the EcoRI and EcoRV sites of pEF1/Myc-His B (Invitrogen Cat.#V92120). The cDNA encoding amino acids 1-386 of human FGFR1c (GenBankAccession number NM_023106) was cloned into the BamHI and NotI sites ofpEF6/Myc-His B (Invitrogen, Cat. number V96220). In both constructs aKozak sequence (CACC) was included immediately before the start codonand a stop codon was added before the Myc-His tag in the vector. Murinepre-B 300-19 cells were co-transfected with β-klotho and FGFR1c plasmidsby electroporation using the Amaxa Nucleofector device and Nucleofectorkit (Lonza, Cat #VCA-1003). Stable clones were selected using 1 mg/mlGeneticin (Invitrogen, Cat #10131) and 8 ug/ml Blasticidin (Invitrogen,Cat #46-1120) for 3 weeks.

Example 2: Preparation of FGFR β-Klotho HEK293 Cells for Use in CellAssays

To test the binding specificity, functional activity, or orthologcross-reactivity of 3-klotho antibodies, HEK293 cells stably expressinghuman FGFR1c_β-klotho, human FGFR2c_β-klotho, human FGFR3c_β-klotho,human FGFR4_β-klotho, or cynomolgus monkey FGFR1c_β-klotho weregenerated using standard Lipofectamine 2000 transfection and cell cloneselection methods.

The following mammalian expression plasmids encoding full-length humanβ-klotho (NM_175737), human FGFR1c (NM_023106), human FGFR2c(NP_001138387), human FGFR3c (NP_000133), or human FGFR4 (NP_998812)cDNAs were used: for cynomolgus monkey β-klotho, the full-lengthsequence was PCR amplified from cynomolgus monkey adipose tissue cDNA(BioChain, Cat. #C1534003-Cy) with primers based on the human and rhesusmonkey β-klotho sequences, and cloned. The cynomolgus monkey FGFR1c cDNAwas cloned from cynomolgus monkey adipose tissue cDNA (BioChain, Cat.#C1534003-Cy) using primers based on the human FGFR1c sequence(#NM_023106) and was shown to be 100% identical at the amino acid levelto human FGFR1c. Hence, the human FGFR1c cDNA construct described abovewas used to make HEK293 cells which stably expressed cynomolgus monkeyβ-klotho and human FGFR1c (#NM_023106) since the human and cynomolgusmonkey FGFR1c amino acid sequences are identical.

Example 3: Measuring FGFR β-Klotho Receptor Activation Using a PERK CellAssay

Standard techniques were used for cell culture and to measurephospho-ERK 1/2 (pERK) levels. Briefly, HEK293 cells stably expressinghuman FGFR1c_β-klotho, human FGFR2c_β-klotho, human FGFR3c_β-klotho,human FGFR4_β-klotho, or cynomolgus monkey FGFR1c_β-klotho weremaintained in DMEM medium (Invitrogen, 11995) containing 10% FBS(Hyclone, SH30071), blasticidin (Invitrogen, A1113902), and Geneticin(Invitrogen, 10131035) at 37° C. in 5% CO₂. Cells were plated into384-well poly-D-lysine-coated plates (BD Biosciences, 354663) andincubated overnight at 37° C. in 5% CO₂, followed by serum-starvation.

Hybridoma supernatants or β-klotho antibodies were diluted in Freestyle293 media and various concentrations of the antibodies were added to theplate. Following incubation, the cells were washed, then lysed withlysis buffer. Cell lysates were transferred to a 384-well assay plate(PerkinElmer, Cat. #6008280) and the AlphaScreen SureFirem pERK 1/2 Kit(Perkin Elmer, TGRES10K) was used to measure phospho-ERK 1/2 levels.Plates were read on the EnVision 2104 multi-label reader (Perkin Elmer)using standard AlphaScreen settings. Dose-response data was graphed aspERK activity fold over basal versus protein concentration to determineEC₅₀ values using the equation Y=Bottom+(Top-Bottom)/(1+10{circumflexover ( )}((Log EC₅₀−X)×HillSlope)) and GraphPad Prism 5 Software.

Example 4: Preparation of Monoclonal Antibodies

Anti-human-β-klotho antibodies were generated in Balb/c (JacksonLaboratory strain: BALB/cJ) or Bcl2 22 wehi (Jackson Laboratory strain:C.Cg-Tg(BCL2)22Wehi/J) mice by whole cell immunizations essentially asdescribed in Dreyer et al (2010) (Dreyer A M et. al. (2010) BMCBiotechnology 10:87).

Briefly, 1×10⁷ human FGFR1c_β-klotho_300.19 cells were injected intoBalb/c mice six times at 10 to 30 day intervals. The first whole cellinjections were done with Freund's Complete Adjuvant (Sigma-AldrichF5881). Cells and adjuvant were not mixed, but injected separately intwo close, but distinct subcutaneous sites. These were followed later byintraperitoneal injections of the same cells with either Sigma AdjuvantSystem (Sigma-Aldrich S6322) or without adjuvant.

Using Bcl2 22 wehi mice, 1×10⁷ human FGFR1c_β-klotho_300.19 cells wereinjected into these animals four times at seven day intervals. The firstinjections were done with Freund's Complete Adjuvant (Sigma-AldrichF5881). Cells and adjuvant were injected separately in two sets of twoclose, but distinct subcutaneous sites Subsequent injections of cellswere done subcutaneously without adjuvant.

Immune responses in the immunized mice were measured by afluorescence-activated cell sorting (FACS) assay. Serum from theimmunized mice diluted 1,000- or 10,000-fold was used to stain humanFGFR1c_β-klotho_HEK and human FGFR1c_β-klotho_300.19 cells, followed byan allophycocyanin (APC) secondary anti-murine IgG detection antibody(Jackson ImmunoResearch Cat #115-136-071). Fluorescence was read on aBecton Dickinson LSRII or Foressa flow cytometer. Four mice with thehighest titer were chosen for electrofusions.

Example 5: Hybridoma Screening, Subcloning, and Selection

2×10⁸ spleenoctyes and 5×10⁷ fusion partner FO cells (ATCC, CRL-1646)were washed in Cytofusion Medium (LCM-C, Cyto Pulse Sciences) and fusedusing a Hybrimune Waveform Generator (Cyto Pulse Sciences, modelCEEF-50B) according to manufacturer's specification with a peak pulse of600 volts. Cells were plated into 384 well plates at a calculateddensity of 3,000 FO cells per well and cultured in HAT selection media(Sigma-Aldrich Cat. H0262).

The primary screen was performed using a high throughput FACS platform(Anderson, Paul. Automated Hybridoma Screening, Expansion, Archiving andAntibody Purification. In: 3rd Annual 2014 SLAS Conference. Jan. 18-22,2014, San Diego, Calif.). Briefly, hybridoma supernatants were incubatedwith human FGFR1c_β-klotho stably expressing and non-expressing celllines and antibody binding was determined with an anti-murine IgG-APCsecondary antibody (Jackson ImmunoReseach Cat #115-136-071).

Antibodies from each hybridoma supernatant were tested for bindingsimultaneously against four barcoded cell lines: 300.19 parental cells,human_FGFR1c_β-klotho_300.19 cells, parental HEK 293 cells, and humanFGFR1c-β-klotho_HEK 293 cells. 348 hits were chosen in the primaryscreen. Primary hits were expanded in 96-well plates and binding wasconfirmed again on human FGFR1c_β-klotho_HEK 293 cells by FACS, yielding122 confirmed hits. HAT (hypoxanthine-aminopterin-thymidine)media-containing supernatants of 115 FACS binding reconfirmed hits wereprofiled for cell activation of the human FGFR1c_β-klotho receptorcomplex using the phospho-ERK 1/2 assay described in Example 2.

Hybridomas with the highest phospho-ERK 1/2 cell activity in theresupernatants were expanded and IgGs were purified from theirsupernatants. Purified IgGs from 74 hybridomas were profiled for cellactivation of the human FGFR1c_β-klotho receptor complex using thephospho-ERK 1/2 assay described in Example 2. IgGs from hybridomas withthe best potency for phospho-ERK 1/2 activation of the humanFGFR1c_β-klotho receptor complex were profiled for orthologcross-reactivity to the cynomolgus monkey FGFR1c_β-klotho receptorcomplex and selectivity for the human FGFR2c_β-klotho and humanFGFR3c_β-klotho receptor complexes using the phospho-ERK 1/2 assaydescribed in Example 2. On the basis of these profiling results, thehybridoma clones, 99G09 and 127F19, were selected the for furtherprofiling.

To evaluate 99G09 and 127F19 signalling in cells expressing α-klotho,HEK293 cells were transfected with α-klotho, Egr1-luciferase and Renillaluciferase. Briefly, HEK293 cells were cultured in DMEM, 10% FBS andplated at 30000 cells/well and transfected with Klotho, Egr-1-luc andTK-Rennila using Lipofectamine 2000. Next day, FGF23, FGF21, 99G09, and127F19 were diluted to the indicated concentration in DMEM supplementedwith 0.1% FBS and added to transfected cells overnight. Luciferaseactivities were detected by Dual-Glo luciferase assay kit (Promega,E2920) according to manufacturer's instruction. As expected, FGF23,which requires α-klotho expression for its signaling, showed strongluciferase expression. However, neither FGF21, 99G09, or 127F19 showedany significant luciferase expression, suggesting that α-klotho does notact as co-receptor for FGF21 or these FGF21 mimetic antibodies.

Example 6: Humanization and Affinity-Maturation of Monoclonal Antibodies

Humanization

The process of humanization is well described in the art (Jones P T etal. (1986) Nature 321: 522-525; Queen C et al. (1989) PNAS USA 86:10029-10033; Riechmann L et al. (1988) Nature 33:323-327; Verhoeyen M etal. (1988) Science 239: 1534-1536). The term humanization describes thetransfer of the antigen-binding site of a non-human antibody, e.g. amurine derived antibody, to a human acceptor framework, e.g. a humangermline sequence (Retter I et al. (2005). Nucleic Acids Res.33:D671-D674.).

The main rationale for humanizing an antibody is seen in minimizing therisk of developing an immunogenic response to the antibody in humans(Rebello P R et al. (1999) Transplantation 68: 1417-1420). Theantigen-binding site comprises the complementary determining regions(CDRs) (Chothia C and Lesk A M (1987) Journal of Molecular Biology 196:901-917; Kabat E et al. (1991) Anon. 5th Edition ed; NIH Publication No.91: 3242) and positions outside the CDR, i.e. in the framework region ofthe variable domains (VL and VH) that directly or indirectly affectbinding. Framework residues that may directly affect binding can, forexample, be found in the so called “outer” loop region located betweenCDR2 and CDR3. Residues that indirectly affect binding are for examplefound at so called Vernier Zones (Foote J, Winter G. (1992) Journal ofMolecular Biology 224:4 87-499). They are thought to support CDRconformation. Those positions outside the CDRs are taken into accountwhen choosing a suitable acceptor framework to minimize the number ofdeviations of the final humanized antibody to the human germlineacceptor sequence in the framework regions.

Sequence Optimization Affinity Maturation

Certain amino acid sequence motifs are known to undergopost-translational modification (PTM) such as glycosylation (i.e. NxS/T,x any but P), oxidation of free cysteines, deamidation (e.g. NG) orisomerization (e.g. DG). If present in the CDR regions, those motifs areideally removed by site-directed mutagenesis in order to increaseproduct homogeneity.

The process of affinity maturation is well described in the art. Amongmany display systems, phage display (Smith G P, 1985, Filamentous fusionphage: novel expression vectors that display cloned antigens on thevirion surface. Science 228:1315-1317) and display on eukaryotic cellssuch as yeast (Boder E T and Wittrup K D, 1997, Yeast surface displayfor screening combinatorial polypeptide libraries. Nature Biotechnology15: 553-557) seem to be the most commonly applied systems to select forantibody-antigen interaction. Advantages of those display systems arethat they are suitable for a wide range of antigens and that theselection stringency can be easily adjusted. In phage display, scFv orFab fragments can be displayed and in yeast display full-length IgG inaddition. Those commonly applied methods allow selection of a desiredantibody variants from larger libraries with diversities of more than10E7. Libraries with smaller diversity, e.g. 10E3, may be screen bymicro-expression and ELISA. Non-targeted or random antibody variantlibraries can be generated for example by error-prone PCR (Cadwell R Cand Joyce G F, 1994, Mutagenic PCR. PCR Methods Appl. 3: S136-S140) andprovide a very simple, but sometimes limited approach. Another strategyis the CDR directed diversification of an antibody candidate. One ormore positions in one or more CDRs can be targeted specifically usingfor example degenerated oligos (Thompson J et al., 1996, Affinitymaturation of a high-affinity human monoclonal antibody against thethird hypervariable loop of human immunodeficiency virus: use of phagedisplay to improve affinity and broaden strain reactivity. J. Mol. Biol.256: 77-88) trinucloetide mutagenesis (TRIM) (Kayushin A L et al., 1996,A convenient approach to the synthesis of trinucleotidephosphoramidites—synthons for the generation of oligonucleotide/peptidelibraries. Nucleic Acids Res. 24: 3748-3755) or any other approach knownto the art.

Generation of Expression Plasmids

DNA sequences coding for humanized VL and VH domains were ordered atGeneArt (Life Technologies Inc. Regensburg, Germany) including codonoptimization for homosapiens. Sequences coding for VL and VH domainswere subcloned by cut and paste from the GeneArt derived vectors intoexpression vectors suitable for secretion in mammalian cells. The heavyand light chains were cloned into individual expression vectors to allowco-transfection. Elements of the expression vector include a promoter(Cytomegalovirus (CMV) enhancer-promoter), a signal sequence tofacilitate secretion, a polyadenylation signal and transcriptionterminator (Bovine Growth Hormone (BGH) gene), an element allowingepisomal replication and replication in prokaryotes (e.g. SV40 originand ColEl or others known in the art) and elements to allow selection(ampicillin resistance gene and zeocin marker).

Expression and Purification of Humanized Antibody Candidates

Human Embryonic Kidney cells constitutively expressing the SV40 large Tantigen (HEK293-T ATCC11268) are one of the preferred host cell linesfor transient expression of humanized and/or optimized IgG proteins.Transfection is performed using PEI (Polyethylenimine, MW 25.000 linear,Polysciences, USA Cat. No. 23966) as transfection reagent. The PEI stocksolution is prepared by carefully dissolving 1 g of PEI in 900 ml cellculture grade water at room temperature (RT).

Purification was performed in two steps, from which humanized andaffinity-matured mAbs were generated from mouse hybridomas. NOV001 isthe humanized and affinity-matured mAb derived from the mouse hybridoma99G09. NOV002 is the humanized and affinity-matured mAb derived from themouse hybridoma 127F19. The IgG1 L234A/L235A (LALA) or IgG1K D265A/P329A(DAPA) isotypes were selected as preventative measures to reduce theantibody's ability to promote antibody-dependent cell-mediatedcytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) (HezarehM et al. (2001) Journal of Virology 75: 12161-12168). NOV001 is the IgG1(LALA) isotype and NOV003 is the IgG1 (DAPA) isotype of the same mAb.NOV002 is IgG1 (LALA) isotype and NOV004 is the IgG1 (DAPA) isotype ofthe same mAb.

Example 7: In Vitro Charactionization of Monoclonal Antibodies

The SET assay as described in Example 5 was used to estimate the KDs ofmAbs to human β-klotho. NOV001, NOV002, and NOV004 have KDs of about 42pM, 8 pM, and 9 pM, respectively (FIG. 1 ). The pERK assay as describedin Example 3 was used to profile mAbs for FGFR_β-klotho receptoractivity. NOV002 activated the human and cynomolgus monkeyFGFR1c_β-klotho receptor complex with EC50s of about 5 nM and 37 nM,respectively (FIG. 2 ). NOV004 activated the human and cynomolgus monkeyFGFR1c_β-klotho receptor complex with an EC50s of about 6 nM and 40 nM,respectively (FIG. 2 ). NOV002 and NOV004 did not activate humanFGFR2c_β-klotho, FGFR3c_β-klotho, or FGFR4_β-klotho receptor complexes(FIG. 3 ). The mAbs were profiled for FGF23 activity as described inExample 5. NOV002 and NOV004 did not exhibit FGF23 activity (FIG. 4 ).The mAbs were profiled for cross-reactivity to the mouse FGFR_β-klothoreceptor complex as described in Example 5. The mAbs NOV002 and NOV004did not lead to glucose-uptake by mouse 3T3L1 adipocyte cells (FIG. 5 ).

Example 8: Epitope Mapping by Hydrogen-Deuterium Exchange of Humanβ-Klotho Extracellular Domain with NOV001 and NOV002

Hydrogen-deuterium exchange (HDx) in combination with mass spectrometry(MS) (Woods V L et al. (2001) High Resolution, High-Throughput AmideDeuterium Exchange-Mass Spectrometry (DXMS) Determination of ProteinBinding Site Structure and Dynamics: Utility in Pharmaceutical Design.J. Cell. Biochem. Supp.; 84(37): 89-98) was used to map the putativebinding sites of NOV001 and NOV002 on human β-klotho extracellulardomain (ECD) (52-997aa). In HDx backbone amide hydrogens of proteins arereplaced by deuterium. This process is sensitive to proteinstructure/dynamics and solvent accessibility and, therefore, able toreport on locations that undergo a decrease in deuterium uptake uponligand binding. The goal of these experiments was to identify thepotential epitopes and understand the dynamics of human β-klotho ECDwhen bound to NOV001 or NOV002. Changes in deuterium uptake aresensitive to both direct binding and allosteric events.

HDx-MS experiments were performed using methods similar to thosedescribed in the literature (Chalmers M J et al. (2006) Probing proteinligand interactions by automated hydrogen/deuterium exchange massspectrometry. Anal. Chem.; 78(4): 1005-1014). The experiments wereperformed on a Waters HDx-MS platform that includes a LEAP autosampler,nanoACQUITY UPLC, and Synapt G2 mass spectrometer. The deuterium bufferused to label the protein backbone of human β-klotho ECD (52-997aa) wasD-PBS, pH 7.2; the overall percentage of deuterium in the solution was95%. The protein human β-klotho (52-997aa) was ordered from R&D System(5889-KB-050). The protein was dialyzed against PBS buffer pH 7.2overnight prior to HDx-MS analysis. For human β-klotho ECD (52-997aa)deuterium labeling experiments in the absence of antibody, 300 pmol ofhuman β-klotho ECD (52-997aa), volume of 13 μl, was diluted using 100 μlof the deuterium buffer (95% deuterium) in a chilled tube and incubatedfor 15 minutes on a rotator at 4° C. The labeling reaction was thenquenched with 100 μl of chilled quench buffer at 2° C. for five minutesfollowed by injected onto the LC-MS system for automated pepsindigestion and peptide analysis.

For human β-klotho ECD (52-997 aa) deuterium labeling experiments in thepresence of NOV001 or NOV002, 350 pmol of NOV001 or NOV002 was firstimmobilized on Thermo Protein G Plus beads and cross-linked with DSS(disuccinimidyl suberate). To perform the labeling experiments, theantibody beads containing 350 pmol antibody were incubated with 300 pmolhuman β-klotho ECD (52-997aa) for 20 minutes at 4° C. After 20 minutesthe beads were washed with 200 μl of PBS buffer. Then 200 μl of chilleddeuterium buffer (84.1% deuterium) was added and the complex wasincubated for 15 minutes at 4° C. After 15 minutes, the deuterium bufferwas spun out and the labeling reaction was quenched with 200 μl ofchilled quench buffer on ice for 4.5 minutes. After spinning the samplefor 30 seconds in a centrifuge, the quenched solution was injected ontothe LC-MS system for automated pepsin digestion and peptide analysis.

All deuterium exchange experiments were quenched using 0.5 M TCEP(tris(2-carboxyethyl)phosphine) and 3 M urea (pH=2.6). After quenching,the exchanged antigen was subjected to on-line pepsin digestion using aPoroszyme Immobilized Pepsin column (2.1×30 mm) at 12° C. followed bytrapping on a Waters Vanguard HSS T3 trapping column. Peptides wereeluted from the trapping column and separated on a Waters BEH C18 1×100mm column (maintained at 1° C.) at a flow rate of 40 μl/min using abinary 8.4 minute gradient of 2 to 35% B (mobile phase A was 99.9% waterand 0.1% formic acid; mobile phase B was 99.9% acetonitrile and 0.1%formic acid).

For human β-klotho ECD (52-997 aa) 82% of the sequence was monitored bythe deuterium exchange experiments as described herein. For differentialexperiments between NOV001 or NOV002 bound and unbound β-klotho ECD, itis informative to examine the change in deuterium incorporation betweenthe two states. In Table 2 a negative value indicates that the NOV001-or NOV002-β-klotho ECD complex undergoes less deuterium uptake relativeto β-klotho ECD alone. A decrease in deuterium uptake can be due toprotection of the antigen from exchangeable deuterium by the antibody orstabilization of the hydrogen bonding network. In contrast, a positivevalue indicates that the complex undergoes more deuterium uptakerelative to β-klotho ECD alone. An increase in deuterium uptake can bedue to destabilization of hydrogen bonding networks (i.e. localizedunfolding of the protein). When examining the differential change indeuterium exchange between two different states, such as apo β-klothoECD and NOV001- or NOV002-β-klotho ECD complex, an approach is typicallyused to determine if the changes are significant. Typically, as long asthe difference is greater than 0.5 Da, the difference is consideredsignificant (Houde D, et al. (2010) J. Pharma. Sci.; 100(6): 2071-2086).

Table 2 lists the change in deuterium incorporation for the NOV001- orNOV002-β-klotho ECD complex relative to the β-klotho ECD alone. Using a−0.5 Da significant cutoff (Houde D, et al. (2010) The Utility ofHydrogen/Deuterium Exchange Mass Spectrometry in BiopharmaceuticalComparability Studies. J. Pharma. Sci.; 100(6): 2071-2086). Thefollowing peptides are significantly protected in the NOV001-β-klothoECD complex: 245-266, 344-349, 421-429, 488-498, 509-524, 536-550,568-576, 646-670, 696-700, 773-804, 834-857, and 959-986 aa. Thefollowing peptides are significantly protected in the NOV002-β-klothoECD complex: 246-265, 343-349, 421-429, 488-498, 509-524, 536-550,568-576, 646-669, 773-804, 834-857, and 959-986 aa.

By comparing the protection values of overlapping peptides one canfurther improve the resolution of the deuterium exchange data. Forexample, the peptide 329-342aa is protected by only −0.51 Da in theNOV001-β-klotho ECD complex, while the larger peptide 330-348 aa isprotected by −1.48 Da. Hence, one can deduce that the significantlyprotected region of the larger peptide must be the region 343-347aa.Performing this analysis across the data reveals that the regions,246-265, 536-550, 834-857 and 959-986 aa are the most strongly protectedupon either NOV002 or NOV001 binding to β-klotho ECD (e.g., to saidregions within SEQ ID NO:262).

Moreover, overall many regions, besides the most strongly protectedregions mentioned earlier, protected by NOV001 are also protected inNOV002. These regions of protection are spread across many regions inthe linear sequence space although there are more regions of protectiontowards the C-terminal side of the ECD. In contrast, there are fewregions that differentiate between the two antibodies. For NOV001 tworegions are uniquely protected: 646-670 aa and 697-700 aa, and forNOV002 one region, 646-689 aa, is uniquely protected. Overall,differentiating the epitopes of NOV002 from NOV001 using HDx-MS alone onβ-klotho ECD was challenging. HDx data suggest that the epitopes arequite similar.

Table 2 shows the effect of NOV001 and NOV002 binding on the deuteriumincorporation human β-klotho (53-997aa). For each peptide detected bymass spectrometry, the change in deuterium incorporation (in Daltons)for the NOV001-β-klotho complex relative to β-klotho ECD alone andNOV002-β-klotho complex relative to N-klotho ECD alone is shown.

TABLE 2HDx-MS peptide coverage of the human β-klotho extracellular domain (52-997aa)Change in Change in Deuterium Deuterium SEQ ID Start End IncorporationIncorporation Sequence NO: Position Position (N0V001) (N0V002) FSGDGRAIW85 53 61 −0.37 −0.36 FLYDTFPKNFF 86 76 86 −0.26 −0.50 FFWGIGT 87 85 910.22 0.10 FFWGIGTGAL 88 85 94 −0.10 −0.12 GIGTGALQ 89 88 95 −0.15 −0.12QVEGSWKKDGKGPSIWDHF 90 95 113 −0.77 −0.79 LEKDLSA 91 134 140 −0.16 −0.19LEKDLSAL 92 134 141 −0.12 −0.20 SALDFIGVSFYQFSISWPRLFPDGIVTVAN 93 139168 −0.23 −0.03 SISWPRL 94 152 158 −0.07 −0.07 SISWPRLFPDGIVT 95 152 165−0.28 −0.34 FPDGIVT 96 159 165 −0.19 −0.24 VANAKGLQ 97 166 173 −0.66−0.70 VANAKGLQY 98 166 174 −0.67 −0.67 LVLRNIEPIVT 99 182 192 −0.02−0.07 VLRNIEPIVT 100 183 192 −0.09 −0.05 RNIEPIVT 101 185 192 −0.11−0.12 RNIEPIVTL 102 185 193 −0.07 −0.08 LYHWDLPLAL 103 193 202 −0.05−0.11 IFNDYAT 104 217 223 −0.02 −0.04 FNDYATYC 105 218 225 −0.16 −0.03CFQMFGDRVKY 106 225 235 −0.07 −0.10 FQMFGDRVK 107 226 234 −0.10 −0.11FQMFGDRVKY 108 226 235 −0.03 −0.05 VAWHGYGTGMHAPGEKGNL 109 245 263 −0.84−0.82 AWHGYGTGMHAPGEKGNL 110 246 263 −0.84 −0.83 WHGYGTGMHAPGEKGNL 111247 263 −1.14 −0.97 WHGYGTGMHAPGEKGNLAA 112 247 265 −1.72 −1.56HGYGTGMHAPGEKGNL 113 248 263 −0.71 −0.72 YTVGHNLIKA 114 267 276 −0.21−0.24 YTVGHNLIKAHSKVWHNYNTHFRPHQKGW 115 267 295 0.13 0.01YTVGHNLIKAHSKVWHNYNTHFRPHQKGWL 116 267 296 −0.23 −0.26VGHNLIKAHSKVWHNYNTHFRPHQKGWL 117 269 296 −0.29 −0.35GHNLIKAHSKVWHNYNTHFRPHQKGWL 118 270 296 −0.16 −0.16LIKAHSKVWHNYNTHFRPHQKGWL 119 273 296 −0.24 −0.27 SITLGSH 120 297 303−0.12 −0.12 SITLGSHWIEPNRSENTMD 121 297 315 −0.22 −0.28 IFKCQQSMV 122316 324 0.13 0.09 FKCQQSM 123 317 323 −0.07 −0.09 KCQQSMV 124 318 324−0.16 −0.20 FANPIHGDGDYPEG 125 330 343 −0.58 −0.51 FANPIHGDGDYPEGMRKKL126 330 348 −1.12 −1.18 FANPIHGDGDYPEGMRKKLF 127 330 349 −0.92 −1.00LPIFSEAEKHEMRGT 128 352 366 −0.99 −1.03 PIFSEAEKHEMRGTAD 129 353 368−0.69 −0.67 PIFSEAEKHEMRGTADF 130 353 369 −0.48 −0.47 SEAEKHEMRGTADF 131356 369 −0.54 −0.54 AEKHEMRGTADF 132 358 369 −0.29 −0.32 EKHEMRGTADF 133359 369 −0.26 −0.28 FAFSFGPNNF 134 370 379 −0.05 −0.10 FAFSFGPNNFKPLNT135 370 384 −0.51 −0.62 FGPNNFKPLNT 136 374 384 −0.50 −0.48 FGPNNFKPLNTM137 374 385 −0.47 −0.59 NLREALN 138 395 401 −0.16 −0.18 IKLEYNNPRIL 139403 413 −0.19 −0.21 EYNNPRIL 140 406 413 −0.02 −0.05 FTDSRVKTEDTTA 141420 432 −1.06 −1.02 DSRVKTEDTTA 142 422 432 −0.94 −0.91 DTTAIYMMKNF 143429 439 −0.58 −0.48 YMMKNFL 144 434 440 −0.05 −0.01 MMKNFLSQVLQA 145 435446 −0.06 −0.07 MKNFLSQVLQA 146 436 446 −0.02 −0.07 QAIRLDE 147 445 451−0.05 −0.03 DEIRVFGYTA 148 450 459 −0.30 −0.26 IRVFGYTA 149 452 459−0.10 −0.09 IRVFGYTAWSL 150 452 462 −0.21 −0.22 YTAWSLL 151 457 463−0.09 −0.06 DGFEWQDA 152 464 471 0.00 −0.12 FEWQDAYT 153 466 473 −0.09−0.13 YTIRRGLF 154 472 479 −0.09 −0.11 TIRRGLF 155 473 479 −0.12 −0.13NSKQKERKPKSSAHY 156 484 498 −1.00 −0.91 NSKQKERKPKSSAHYYKQIIRE 157 484505 −0.75 −0.70 NSKQKERKPKSSAHYYKQIIRENG 158 484 507 −0.80 −0.76NSKQKERKPKSSAHYYKQIIRENGF 159 484 508 −0.65 −0.66NSKQKERKPKSSAHYYKQIIRENGFS 160 484 509 −1.11 −1.02 SKQKERKPKSSAHYYKQIIRE161 485 505 −0.72 −0.69 YKQIIRENG 162 499 507 −0.42 −0.43FSLKESTPDVQGQFPCD 163 508 524 −0.79 −0.89 SLKESTPDVQGQFPCD 164 509 524−1.02 −1.22 LKESTPDVQGQFPCD 165 510 524 −0.98 −1.07 FSWGVTE 166 525 531−0.20 −0.19 SVLKPESVASSPQFSDPHL 167 532 550 −1.74 −1.87 KPESVASSPQFSDPHL168 535 550 −1.74 −1.74 VRLKTRPAQC 169 567 576 −1.37 −1.35 FVNIKKQLEM170 579 588 −0.91 −0.90 VNIKKQLEM 171 580 588 −0.68 −0.68 VNIKKQLEML 172580 589 −0.72 −0.74 NIKKQLEM 173 581 588 −0.41 −0.51 LARMKVTHYR 174 589598 −0.12 −0.08 LARMKVTHYRF 175 589 599 −0.16 −0.16 ARMKVTHYRF 176 590599 −0.14 −0.12 ALDWASVL 177 600 607 −0.08 −0.11 YRCVVSEG 178 623 630−0.23 −0.22 VVSEGLKLGISA 179 626 637 −0.13 −0.10 GLKLGISAM 180 630 638−0.10 −0.05 LKLGISA 181 631 637 −0.06 −0.06 LKLGISAM 182 631 638 −0.04−0.06 ISAMVTLYYPT 183 635 645 −0.22 −0.18 LYYPTHAHLGLPEPLL 184 641 656−0.55 −0.86 YYPTHAHLGLPEPLL 185 642 656 −0.63 −0.83YYPTHAHLGLPEPLLHADGWLNPSTAEA 186 642 669 −1.29 −1.04 HADGWLNPSTAEA 187657 669 −0.83 −0.61 HADGWLNPSTAEAF 188 657 670 −0.62 −0.48 AFQAYAGL 189669 676 −0.11 −0.09 FQAYAGLC 190 670 677 −0.08 −0.09 QAYAGLC 191 671 677−0.13 −0.15 QAYAGLCF 192 671 678 −0.12 −0.16 CFQELGD 193 677 683 −0.18−0.20 CFQELGDL 194 677 684 0.01 −0.07 WITINEPNRL 195 688 697 −0.70 −0.46WITINEPNRLSD 196 688 699 −0.80 −0.54 WITINEPNRLSDI 197 688 700 −1.24−0.68 ITINEPNR 198 689 696 −0.68 −0.40 VAHALAWRL 199 717 725 −0.12 −0.11AHALAWRL 200 718 725 −0.06 −0.06 HALAWRL 201 719 725 −0.14 −0.13YDRQFRPSQRGAVS 202 726 739 −0.48 −0.48 YDRQFRPSQRGAVSL 203 726 740 −0.48−0.43 DRQFRPSQRGAVS 204 727 739 −0.60 −0.58 SLHADWAEPANPYADSHWRAAERF 205741 764 −0.45 −0.68 HADWAEPANPYADSHW 206 743 758 −0.34 −0.43HADWAEPANPYADSHWRA 207 743 760 −0.69 −0.78 HADWAEPANPYADSHWRAAERF 208743 764 −0.73 −0.92 WAEPANPYADSHWRAAERF 209 746 764 −0.74 −0.92PANPYADSHWRAAERF 210 749 764 −0.33 −0.51 PYADSHWRAAERF 211 752 764 −0.51−0.68 FAEPLFKTGDYPAA 212 772 785 −0.66 −0.68 FAEPLFKTGDYPAAM 213 772 786−0.87 −0.83 KTGDYPAAM 214 778 786 −0.84 −0.81 REYIASKHRRGLSSSAL 215 787803 −1.21 −1.00 REYIASKHRRGLSSSALPRL 216 787 806 −1.45 −1.25YIASKHRRGLSSSAL 217 789 803 −1.22 −0.98 PRLTEAE 218 804 810 −0.53 −0.58PRLTEAERRLLKGTVDF 219 804 820 −0.24 −0.33 AERRLLKGTVDF 220 809 820 −0.24−0.24 ERRLLKGTVDF 221 810 820 −0.34 −0.34 RRLLKGTVDF 222 811 820 −0.21−0.23 CALNHFTTRF 223 821 830 −0.28 −0.29 VMHEQLAGSRYDSDRD 224 831 846−1.32 −1.07 VMHEQLAGSRYDSDRDIQF 225 831 849 −1.08 −0.97HEQLAGSRYDSDRDIQF 226 833 849 −1.93 −1.88 AGSRYDSDRD 227 837 846 −0.99−0.85 AGSRYDSDRDIQF 228 837 849 −0.64 −0.55 GSRYDSDRDIQF 229 838 849−0.76 −0.69 LQDITRLSSPTR 230 850 861 −1.28 −1.28 LQDITRLSSPTRL 231 850862 −1.69 −1.70 ITRLSSPTRL 232 853 862 −1.09 −1.06 TRLSSPTR 233 854 861−1.00 −0.91 SPTRLAV 234 858 864 −0.19 −0.20 SPTRLAVIPWGVRKL 235 858 872−0.13 −0.18 AVIPWGVRKL 236 863 872 −0.43 −0.60 AVIPWGVRKLLRWVRRNYGDM 237863 883 −0.12 −0.26 AVIPWGVRKLLRWVRRNYGDMD 238 863 884 −0.23 −0.42AVIPWGVRKLLRWVRRNYGDMDI 239 863 885 −0.01 −0.20 IPWGVRK 240 865 871−0.51 −0.35 IPWGVRKLLRWVRRNYGDMD 241 865 884 −0.23 −0.37 LRWVRRNYGDMDI242 873 885 −0.08 −0.11 IYITASGIDDQALED 243 885 899 −0.62 −0.91YITASGIDDQAL 244 886 897 −0.30 −0.21 YITASGIDDQALEDDRLRKYYLGKY 245 886910 −0.90 −1.02 EDDRLRKYYLGKY 246 898 910 −0.55 −0.62 DRLRKYYLGKY 247900 910 −0.35 −0.35 DRLRKYYLGKYLQE 248 900 913 −0.44 −0.50 IDKVRIKG 249920 927 −0.41 −0.40 IDKVRIKGY 250 920 928 −0.37 −0.32 IDKVRIKGYYA 251920 930 −0.44 −0.42 IDKVRIKGYYAF 252 920 931 −0.28 −0.30 DKVRIKGYYA 253921 930 −0.45 −0.43 KVRIKGYYA 254 922 930 −0.34 −0.35 KVRIKGYYAF 255 922931 −0.40 −0.40 FKLAEEKSKPRFGF 256 931 944 −0.83 −0.75 FKLAEEKSKPRFGFF257 931 945 −0.62 −0.59 KLAEEKSKPRFGFF 258 932 945 −0.75 −0.74 FKAKSSIQF259 949 957 −0.89 −0.88 YNKVISSRGFPFENSSSRCSQTQE 260 958 981 −1.64 −1.62SSRCSQTQENTECT 261 973 986 −1.95 −2.01

Example 9: Pharmacokinetic Profiles of Monoclonal Antibodies in Rat

Animals and Maintenance Conditions

Animal care and husbandry were provided according to the Guide for theCare and Use of Laboratory Animals (Institute of Laboratory AnimalResources, National Research Council). All procedures were governed bythe standards set forth by the US Department of Health and HumanServices and performed according to protocol CV9054 approved by theNovartis East Hanover Animal Care and Use Committee. Male,Sprague-Dawley rats (n=3/group) were housed in solid-bottom cages on arack equipped to automatically provide water ad libitum, maintained on a12 hr light/dark cycle (6 am to 6 pm), and given free access to standardrodent chow (Harlan-Teklad; Frederick, Md.; cat #8604). The vivarium wasmaintained between 68 and 76° F. with 30 to 70% humidity.

NOV002 or NOV004 Preparation and Dosing

Stock solutions of NOV002 (12.00 mg/mL) and NOV004 (16.0 mg/mL) in 20 mMHistidine buffer (pH 6.0) were shipped frozen, stored at −80° C., andthawed under refrigeration prior to use. On the morning of dosing, bothantibodies were diluted to 2 mg/mL in 20 mM Histidine buffer andappropriate volumes were drawn into dosing syringes (5 mL/kg) and keptat room temperature until administration. Animals were placed in tuberestrainers and administered either NOV002 or NOV004 via intravenous(IV) injection into the tail vein (10 mg/kg).

Blood Sample Collection

Blood samples were collected on day −3 (Baseline), day 0 (1 and 6 hpost-dose), and days 1, 2, 4, 7, 14, 21, and 28 post-dose. All timepoints were timed from the end of administration of the dose given onday 0. At each timepoint, approximately 0.2 mL (200 μL) of blood wascollected into BD Microtainer collection/separator tubes with EDTA(Becton, Dickinson, and Company; Franklin Lakes, N.J.; cat #365973).Pressure was applied with gauze to stop the bleeding. Samples werecentrifuged for 10 min at 20,817×g, and then ˜100 pL plasma wastransferred to 0.2 mL Thermo-strip tube (Thermo-Scientific; Pittsburgh,Pa.; cat #AB-0451) and frozen at 80° C. Rats were returned to their homecage after each collection.

Measurement of Plasma Total NOV002 or NOV004 Concentrations

Human IgG (i.e. NOV002 or NOV004) in rat plasma was quantified using acustom sandwich immunoassay with a mouse anti-human-IgG monoclonalantibody (R10Z8E9) as capture antibody and a goat anti-human-IgG with anHRP label as detection antibody. The capture antibody (2 μg/mL in PBS,30 μL/well) was coated on 384-well, white, microtiter plates (GreinerBio-One; Monroe, N.C.; cat no. 781074). The plates were incubatedovernight at room temperature (RT) without shaking. After aspirating thecoating solution without washing, 90 μL of 1× Milk Diluent/Blockingsolution (KPL; Gaithersburg, Md.; cat no. 50-82-01) was added to eachwell and the plates were incubated for 2 h at RT. At the end of theincubation, the solution was aspirated and the plates were stored infoil pouches with desiccant at −80° C.

On the day of the assay, sixteen NOV002 or NOV004 standardconcentrations, ranging from 0.15-600 ng/mL, were prepared by serialdilution in Casein buffer, including a buffer negative control. Allstudy samples were diluted 1:50 manually in Casein buffer and thenserially diluted 5-fold using a Freedom EVO (Tecan; Mannedorf,Switzerland) for a total of five dilutions. The plates were incubatedfor 2 h at RT, with shaking at 300 rpm, and then washed 3 times withphosphate wash buffer (90 μL/well). HRP-labeled goat anti-human-IgG (400ng/mL in Casein buffer, 30 μL/well) was added to each plate and theplates were incubated for 1 h at RT, with shaking at 300 rpm. The plateswere washed 3 times with phosphate wash buffer (90 μL/well), and thenKPL LumiGLO Chemiluminescent Substrate was added (30 μL/well; cat no.54-61-00). Chemiluminescence was read immediately on a SpectraMax M5plate reader (Molecular Devices) at all wavelengths with 50 msintegration time. Human Fc concentrations (ng/mL) in plasma samples wereinterpolated from the NOV002 or NOV004 standard curve, multiplied bydilution factors, and converted to nM concentrations.

Animals exhibited mean C_(max) of 1207 and 1115 nM at 1 h after IVadministration of NOV002 or NOV004, respectively. NOV002 and NOV004exhibit equivalent PK profiles in Sprague-Dawley rats (FIG. 6 ).

Example 10: Study in Obese, Cynomolgus Monkeys

The effects of NOV002 on food consumption, body weight, and plasmabiomarkers in obese cynomolgus monkeys were studied.

Protocols:

Five male cynomolgus monkeys were treated with two subcutaneous (s.c.),1 mg/kg doses of NOV002 administered one week apart (study days 0 and 7)and food consumption, body weights, and plasma biomarkers were assessedfor more than 100 days post-dose. For each dose, animals were restrainedin their home cage, blood samples were collected, and then each animalwas given a subcutaneous dose of 1 mg/kg NOV002. Food consumptionmeasurements started 1 week before the first dose and continued throughthe study. The study diet was weighed prior to feeding and divided intotwo equal portions for each day. The following morning, remaining dietwas collected and weighed. The number of pellets (1 g each) dropped inthe catch pan were counted and added to the weight of the remainingfood. Daily food consumption was calculated as the weight of foodprovided minus food collected. Fruit and vegetable consumption were notmeasured. Non-fed body weights were measured in duplicate three morningsper week (prior to blood collection or dosing) using the dynamic featureon the scale.

Measurement of Plasma NOV002 Concentrations

Human Fc IgG in cynomolgous monkey plasma was quantified using an ELISAbased sandwich immunoassay. Anti-human-IgG mouse IgG1, a mousemonoclonal antibody against human IgG, was used as the capture antibody.White, Greiner, 384-well plates were coated with 2 μg/mL anti-human-IgGmouse IgG1 (30 μL/well) and incubated overnight at room temperature(RT). Coating antibody was aspirated and 1× milk blocker (KPL #50-82-01)was added at 90 μL/well for 2 h at RT. The blocking solution wasaspirated and the plates were stored at −80° C. in plate bags withdesiccant until assay. On the day of the assay, plates and reagents werebrought to RT. Standards were made by diluting the purified IgG from4000 to 16 pM in custom casein sample diluent and including a buffercontrol. Samples were diluted in duplicate 1:50, 1:250, 1:1250, and1:6250 in the same diluent as standards, and then standards, dilutedsamples, and controls were added to the plate for 2 h at RT (workingvolume for all steps was 30 μL/well). Plates were then washed 3 timeswith a phosphate based wash buffer. Horseradish peroxidase (HRP)-labeledanti-human Fc-gamma antibody was added to the plate for 1 hour at RT,and then the plates were washed 3 times with a phosphate-based washbuffer. Chemiluminescent substrate was added to the plate and the platewas immediately read on a luminescence plate reader.

NOV002 standards were assayed in triplicate per plate. Diluted plasmasamples were assayed in duplicate. Unknown samples were interpolatedfrom the IgG standard curve. Curve fitting, back-calculation, %recovery, and interpolation of sample concentrations were performedusing SoftMax Pro Software v5.4.1. Signal generated by the IgG standardswas plotted and fit using a 4-parameter logistical curve-fitting option.Fc concentrations (pM) in plasma samples were interpolated from theNOV002 standard curve and multiplied by dilution factors. The assaylower limit of quantification (LLOQ) was 31.250 pM and the upper limitof quantification (ULOQ) was 4000 pM. LLOQ and ULOQ were defined as thelower and upper standard concentration with 100% recovery±20% and CV<20%and then multiplied by the dilution factors.

Detection of Anti-Drug Antibodies

Plasma samples were diluted 1:5 in LowCross Buffer (Boca Scientific;Boca Raton, Fla.; cat no. 100 500). Reaction Mixture was preparedcontaining 0.6 μg/mL of biotin-labeled NOV002 and 0.6 μg/mL ofdigoxigenin-labeled NOV002 in LowCross Buffer. Diluted plasma (80 μL)was combined with 160 μL of Reaction Mixture in 96-well U-bottom plates(BD Falcon; Billerica, Mass.; cat no. 351177). The edges of the plateswere sealed with Parafilm and the plates were incubated on a shakingplatform at 37° C. for 2 h (150 rpm, protected from light). An aliquotof each mixture (100 μL) was then transferred to duplicate wells ofStreptavidin-coated 96-well plates (Roche; cat no. 11734776001), whichwere first washed 3 times with wash buffer consisting of 1×PBScontaining 0.05% (v/v) Tween-20 (300 μL per well). The plates weresealed and then incubated at RT on a shaking platform for 1 h (300 rpm,protected from light). Plates were washed 3 times with wash buffer (300μL per well), and then 100 μL of anti-digoxigenin peroxidase_POD Fabfragment (Roche; cat no. 11633716001) diluted 1:2500 in LowCross Bufferwere added to each well. The plates were sealed, incubated at RT on ashaking platform for 45 minutes (300 rpm, protected from light), andthen washed 3 times with wash buffer (300 μL per well). TMB OneComponent HRP Microwell Substrate (Bethyl Laboratories; Montgomery Tex.;cat no. E102; 100 μL/well) was added to each well and blue color wasdeveloped for 9-10 min., protected from light. The color reaction wasstopped by adding 100 μL of 0.18 N H2SO4 to each well, the plates wereshaken briefly, and yellow color was measured at OD450.

Measurement of Plasma Glucose Concentrations

Plasma glucose concentrations were measured using an Autokit Glucoseassay (Wako Chemicals; Richmond, Va.; catalog no. 439-90901). A standardcurve was prepared by diluting the calibrator to 500, 200, 100, 50, 20,and 0 mg/dL standards. Assay reagent (300 μL), pre-warmed to 37° C., wasadded to 2 μL of plasma, standards, and control samples in a clear,flat-bottom, 96-well plate (Thermo Scientific; cat no. 269620). Theplate was mixed on a plate shaker for 30 s and then incubated at 37° C.for 5 min. Following a 20 s mix, the plate was read at 505/600 nm usinga Molecular Devices SPECTRAmax PLUS 384 (Sunnyvale, Calif.). Sampleglucose concentrations were calculated by comparing to the standardcurve.

Measurement of Plasma Insulin Concentrations

Plasma insulin concentrations were determined using the Millipore HumanInsulin Specific RIA Kit (Billerica, Mass.; cat no. HI-14K) according tothe manufacturer instructions. Appropriate amounts of assay buffer,standards, or diluted plasma sample were mixed with ¹²⁵I-insulin andanti-insulin antibody in 5 mL, 75×12 mm PP SARSTEDT tubes (catalog no.55.526). The tubes were vortexed, covered, and incubated for 20 h at RT.After the incubation, 1 mL of 4° C. precipitating reagent was added andthe tubes were vortexed and incubated for 30 min. at 4° C. All tubeswere centrifuged for 30 min (3000 rpm at 4° C.), the supernatants weredecanted, and the pellets were counted on a PerkinElmer WIZARD2Automatic Gamma Counter (model no. 2470; PerkinElmer; Waltham, Mass.).Insulin concentrations were calculated by comparing to a standard curvegenerated using known quantities of insulin.

Measurement of Plasma Triglyceride Concentrations

Plasma triglyceride (TG) concentrations were measured using theTriglyceride (GPO) Liquid Reagent set (Pointe Scientific; Canton, Mich.;cat no. T7532-500). Pre-warmed assay reagent (300 μL, 37° C.) was addedto 5 μL of plasma in a clear, flat-bottom, 96-well plate (Thermo FisherScientific; Tewksbury, Mass.; cat no. 269620). The plate was mixed on aplate shaker for 30 s and then placed in an incubator at 37° C. for 5min. Following a 20 s mix, absorbance was measured at 500 nm with aSPECTRAmax PLUS plate reader. TG concentrations were calculated bycomparing to a calibration curve generated using known quantities of aTG standard (Pointe Scientific; cat no. T7531-STD).

Measurement of Plasma Cholesterol Concentrations:

Plasma total cholesterol (TC) was quantified using the Cholesterol(Liquid) Reagent Set, (Pointe Scientific; cat no. C7510-500). Pre-warmedassay reagent (200 μL, 37° C.) was added to 10 μL of plasma in a clear,flat-bottom, 96-well assay plate (Thermo Fisher Scientific; cat no.269620). The plate was mixed on a plate shaker for 30 s and thenincubated at 37° C. for 5 min. Following a 20 s mix, absorbance wasmeasured at 500 nm in a SPECTRAmax PLUS plate reader. Cholesterolconcentrations were calculated by comparing to a calibration curvegenerated using known quantities of a cholesterol standard (StanbioLaboratory; Boerne, Tex.; cat no. 1012-030).

Measurement of Plasma High-Density Lipoprotein CholesterolConcentrations

For determination of high-density lipoprotein (HDL) cholesterolconcentrations, 50 μL of plasma sample was combined with 50 μL ofCholesterol Precipitating Reagent (Wako Chemicals; Richmond, Va.; catno. 431-52501) in a 0.5 mL microcentrifuge tube and vortexed briefly.The tube was placed at room temperature for 10 min and then centrifugedat 2000×g for 10 min at 4° C. Following centrifugation, approximatelyhalf of the supernatant (containing the HDL cholesterol portion of theoriginal plasma sample) was removed and 10 μL was used for thecholesterol assay described above.

Measurement of Plasma β-Hydroxybutyrate Concentrations

Plasma β-hydroxybutyrate (β-HB) concentrations were measured using theβ-Hydroxybutyrate LiquiColor Test kit (Stanbio Laboratory; cat no.2440-058). Assay reagent R1 (215 μL pre-warmed to 37° C.) was added to20 μL of quality control or plasma sample in a clear, flat-bottom,96-well plate (Thermo Fisher Scientific; cat no. 269620). The plate wasmixed on a plate shaker for 30 s and then placed in an incubator at 37°C. for 5 min. Pre-read absorbance was measured at 505 nm in a SPECTRAmaxPLUS plate reader. Assay reagent R2 (35 μL pre-warmed to 37° C.) wasadded to each well, and the plate was again mixed on a plate shaker for30 s and incubated at 37° C. for 5 min. Following a 20 s mix, finalabsorbance was measured at 505 nm from which the pre-read value wassubtracted. β-HB concentrations were calculated by comparing to acalibration curve generated using known quantities of a β-HB calibrator(Wako Diagnostics; Richmond, Va.; cat no. 412-73791).

Statistical Analyses

Statistical analyses were performed using GraphPad Prism (Version 6.05;GraphPad Software; La Jolla, Calif.). Food intake data for each animalwere normalized as a percent of baseline (calculated as the mean of days−6 to 0) and then group means±standard errors of the mean (SEM) werecalculated; each day was compared to day 0 by nonparametric Friedman'stest with Dunn's multiple comparisons post-test. Body weights arepresented as group means±standard errors of the mean (SEM) calculated aspercent of baseline (calculated as the mean of days −7, −5, −3, and 0).Raw body weight and plasma biomarker data were also analyzed bynonparametric Friedman's test with Dunn's multiple comparisonspost-test. P<0.05 was considered significant.

Results

Before dosing NOV002, animals were screened for pre-existing anti-drugantibodies (ADA) and found that all monkeys were ADA negative. Todetermine whether NOV002 could reduce food intake and body weights ofobese monkeys (mean baseline body weight=12.4±0.9 kg, which wascalculated as the mean of days −6 to 0), each animal received twosubcutaneous doses of 1 mg/kg NOV002 one week apart on study days 0 and7. NOV002 significantly decreased food intake compared to baseline, withthe mean peak reduction to ˜44% of baseline occurring on day 32post-dose. Food intake was significantly lower on days 18, 22, 24, 25,26, 29, and 32 post-dose vs day 0 by Friedman test with Dunn'spost-test). All five monkeys exhibited clear decreases in foodconsumption, but the magnitude and duration of the reduced food intakewere variable across the group.

Body weights was also measured throughout the study and a peak mean bodyweight change of −8.9% on day 67 post-dose was observed. Body weight wassignificantly lower on days 37 through 70 post-dose vs day 0 by Friedmantest with Dunn's post-test. Similar to the food intake effects, variableextent and duration of body weight changes were observed followingtreatment with 1 mg/kg NOV002 that were consistent with the food intakeresponses of the individual animals: the monkeys with the greatestreductions in food intake lost the most body weight during the study.

In addition to evaluating the effects of NOV002 on food intake and bodyweights, the effects on plasma biomarkers of lipid and carbohydratemetabolism were tested. NOV002 decreased both plasma TG and TCconcentrations, with a significant difference on day 32 post-dose versusbaseline (mean of days −7, −3, and 0; Friedman test with Dunn'spost-test). Table 3 summarizes all plasma biomarker changes on day 32versus baseline. Compared to baseline, NOV002 did not significantlychange plasma high-density lipoprotein (HDL) cholesterol,O-hydroxybutyrate (β-HB), glucose, or insulin concentrations at anytimepoint during the study, but NOV002 did significantly increase plasmaadiponectin levels on day 35 post-dose.

TABLE 3 NOV002 improved plasma biomarker levels Biomarker Baseline¹ Day32 Mean % Δ² TG (mg/dL) 227 ± 89   93 ± 35*  −53 ± 6 TC (mg/dL) 126 ± 11  96 ± 6*  −22 ± 5 HDL (mg/dL) 53 ± 9   53 ± 5     6 ± 12 β-HB (μM) 53 ±5  115 ± 31   137 ± 77 Glucose (mg/dL) 80 ± 3   76 ± 7  −10 ± 9 Insulin(μU/mL) 271 ± 57  124 ± 12  −40 ± 17 Adiponectin (μg/mL)³  2.9 ± 0.6 6.0 ± 1.8*    94 ± 22 Values represent group means ± SEM. ¹Baselinevalues reflect the mean of days −7, −3, and 0. ²Percent change wascalculated for each individual and then averaged for group mean ± SEM.³Adiponectin levels were measured only in a subset of samples, so dataare from study days 0 and 35. *P < 0.05 vs baseline by nonparametricFriedman test with Dunn's post-test.

ADA formation were also tested at various timepoints (e.g., days 1, 2,3, 4, 7 (prior to dosing and 6 h post-dose), twice weekly from days 11through 81, and then once weekly thereafter) throughout the study. Noneof the monkeys displayed NOV002 ADA in the pre-screen, but three animalsdeveloped ADA to NOV002 during the study. NOV002 PK profiles did notappear to be different between ADA positive and ADA negative animals.

NOV002 effectively lowered food intake and body weight in obese,cynomolgus monkeys. In animals treated with two subcutaneous doses of 1mg/kg NOV002, the following were observed: 8.9% peak body weightreduction on day 67 post-dose, although the reductions in food intakeand body weight varied between individual animals. The body weight losswas accompanied by reduced plasma TG and TC concentrations, showingbeneficial effects of NOV002 on plasma lipid profiles. These dataindicate that NOV002 is effective in reducing food intake, body weight,and plasma TG and TC concentrations; suggesting that NOV002, as well asits variants such as NOV004, other anti-β-klotho antibodies comprisingCDRs of NOV002 or NOV004, and other anti-β-klotho antibodies which bindto the same epitope as NOV002 (e.g., antibodies binding to one or moreof the protected peptides set forth in Table 2) or which competes withNOV002, would be an effective new therapy for metabolic disorders, suchas obesity.

INCORPORATION BY REFERENCE

All references cited herein, including patents, patent applications,papers, text books, and the like, and the references cited therein, tothe extent that they are not already, are hereby incorporated herein byreference in their entirety.

EQUIVALENTS

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The foregoingdescription and examples detail certain preferred embodiments of theinvention and describe the best mode contemplated by the inventors. Itwill be appreciated, however, that no matter how detailed the foregoingmay appear in text, the invention may be practiced in many ways and theinvention should be construed in accordance with the appended claims andany equivalents thereof.

The invention claimed is:
 1. A nucleic acid coding for an isolatedantibody or antigen-binding fragment thereof that specifically binds tothe β-klotho sequence (SEQ ID NO: 262), and said antibody orantigen-binding fragment thereof comprises one of: (i) a heavy chainvariable region comprising three heavy chain CDRs, wherein the threeheavy chain CDRs comprise SEQ ID NOS: 23, 24, and 25, and a light chainvariable region comprising three light chain CDRs, wherein the threelight chain CDRs comprise SEQ ID NOS: 33, 34, and 35; (ii) a heavy chainvariable region comprising three heavy chain CDRs, wherein the threeheavy chain CDRs comprise SEQ ID NOS: 26, 27, and 28, and a light chainvariable region comprising three light chain CDRs, wherein the threelight chain CDRs comprise SEQ ID NOS: 36, 37, and 38; (iii) a heavychain variable region comprising three heavy chain CDRs, wherein thethree heavy chain CDRs comprise SEQ ID NOS: 268, 24, and 25, and a lightchain variable region comprising three light chain CDRs, wherein thethree light chain CDRs comprise SEQ ID NOS: 33, 34, and 35; and (iv) aheavy chain variable region comprising three heavy chain CDRs, whereinthe three heavy chain CDRs comprise SEQ ID NOS: 269, 270, and 271, and alight chain variable region comprising three light chain CDRs, whereinthe three light chain CDRs comprise SEQ ID NOS: 272, 273, and
 35. 2. Thenucleic acid of claim 1, wherein the antibody or antigen-bindingfragment thereof comprises a variable heavy chain sequence (VH)comprising the amino acid sequence of SEQ ID NO: 29 or an amino acidsequence with at least 80%, 90%, 95%, or 97% identity thereof; and avariable light chain sequence (VL) comprising the amino acid sequence ofSEQ ID NO: 39 or an amino acid sequence with at least 80%, 90%, 95%, or97% identity thereof.
 3. The nucleic acid of claim 1, wherein theantibody or antigen-binding fragment thereof comprises a variable heavychain sequence (VH) comprising the amino acid sequence of SEQ ID NO: 29and a variable light chain sequence (VL) comprising the amino acidsequence of SEQ ID NO:
 39. 4. The nucleic acid of claim 1, wherein theantibody or antigen-binding fragment thereof comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 31 or SEQ ID NO: 71 anda light chain comprising the amino acid sequence of SEQ ID NO:
 41. 5. Avector comprising the nucleic acid according to claim
 1. 6. A host cellcomprising the vector of claim
 5. 7. A method of making an antibody orantigen-binding fragment thereof which binds β-klotho, comprising thesteps of: culturing the host cell of claim 6 under conditions suitablefor expression of the antibody or an antigen-binding fragment thereof,wherein the antibody or antigen-binding fragment thereof comprises aheavy chain variable region and a light chain variable region which canbe assembled; and isolating and/or purifying said antibody orantigen-binding fragment thereof from the culture medium.